Methods and compositions related to nucleic acid binding assays

ABSTRACT

Small molecule fluorescent probes for established drug targets such as nucleic acids including DNA and RNA has been developed and disclosed herein. These nucleic acid probes bind to multiple DNA and RNA structures, and to sites crucial for nucleic acid function, such as DNA and RNA major grooves. Displacement of the probes by other binders such as small molecule compounds and/or proteins illicits a fluorescence change in the probe that once detected and analyzed provide binding information of these other binders of interest. Similarly, changes in fluorescence upon binding of the probes to nucleic acid have been applied to screen nucleic acid of different sequence and conformation. The nucleic acid probes and method of uses disclosed herein are advantageously suitable for high-through put screening of libraries of small molecule compounds, proteins, and nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. application Ser.No. 13/939,950, filed Jul. 11, 2013 and claims the benefit of priorityto U.S. Provisional Application No. 61/670,141, filed on Jul. 11, 2012.All applications to which the instant application claims priority areherein incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government Support under Grant 1R15CA125724and R41GM097917 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The subject matter disclosed herein generally relates to nucleic acidprobes and the methods of use. The subject matter disclosed hereinfurther relates to the method of making the nucleic acid probes and usein high through put screening.

BACKGROUND

Fluorescently-tagged aminoglycosides have been synthesized by randomlycoupling amine-reactive fluorescent dyes to amino groups of theseantibiotics [Hamasaki & Rando, 1998; Tok, Cho, & Rando, 1999]. However,they are unsatisfactory for use in high-throughput screens because 1)amino groups critical for high affinity binding of the ribosomal RNAA-site have been modified and 2) the ribosomal RNA A-site binding doesnot change their fluorescence intensity and binding constants can onlybe extracted by monitoring changes in fluorescence anisotropy.

It is desirable to have nucleic acid probes which bind all type ofnucleic acid, such as double stranded DNA, RNA, four stranded DNA/RNA ormajor groove interactors. The disclosed compositions and methods providesolutions to these problems in the art. The disclosed compositions andmethods provide nucleic acid probes that will bind all forms of nucleicacid, and which will bind in such a way that the functional groupsinvolved in DNA interactions are not used to attach the fluorescenttags.

SUMMARY

In accordance with the purposes of the disclosed materials, compounds,compositions, articles, devices, and methods, as embodied and broadlydescribed herein, the disclosed subject matter, in one aspect, relatesto nucleic acid probes and methods for preparing the probes and usingthem. Further, the subject matter disclosed herein relates to assaymethods, including high through-put screening methods using the nucleicacid probes disclosed herein.

Additional advantages will be set forth in part in the description thatfollows, and in part will be obvious from the description, or may belearned by practice of the aspects described below. The advantagesdescribed below will be realized and attained by means of the elementsand combinations particularly pointed out in the appended claims. It isto be understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a design of disclosed probes fornucleic acid targeted screening.

FIG. 2A shows the effect of bacterial A-site oligonucleotide (SEQ IDNo. 1) on the fluorescence emission intensity of a fluorescein-neomycinconjugate (F-neo or 42a) disclosed herein.

FIG. 2B shows the decrease of fluorescence emission intensity of F-neowith increased concentration of bacterial A-site oligonucleotide (SEQ IDNO. 1).

FIG. 3 shows the titration of bacterial A-site oligonucleotide (SEQ IDNO. 1)/fluorescein-neomycin conjugate (F-neo) complex with neomycin.

FIGS. 4A-C show results from a F-neo based RNA binding assay using a96-well format.

FIG. 5 shows the Z′ factor of the displacement of F-neo (42a) fromA-site oligonucleotide (SEQ ID No. 1) by neomycin in 96 well format.

FIG. 6A shows effect of pH on the absorbance spectra offluorescein-neomycin.

FIG. 6B shows absorbance at 490 nm of fluorescein-neomycin as functionof pH.

FIG. 7A shows the effect of A-site rRNA oligonucleotide (SEQ ID NO. 1)on the absorbance spectra of fluorescein-neomycin at pH 7.1.

FIG. 7B shows the absorbance at 490 nm of fluorescein-neomycin:A-sitecomplex as function of pH.

FIG. 8A shows absorbance spectra of fluorescein-neomycin with increasingpH showing the transition between mono-anion and di-anion species.

FIG. 8B shows the total change in absorbance spectra between themono-anion and di-anion species of fluorescein neomycin.

FIG. 8C shows the absorbance maximum plotted as a function of pH withthe resulting inflection point representing the fluorescein phenolicpKa.

FIG. 9 is a plot showing the shift in absorbance maximum plotted as afunction of pH for fluorescein-neomycin with one molar equivalentd(CCCCGGGG)₂, and d(GGGGCCCC)₂ compared to free fluorescein-neomycinresulting in the shift in pKa.

FIG. 10 is a plot representing the change in fluorescence (%) offluorescein-neomycin with 1 equivalent d(GGGGCCCC)₂ and d(CCCCGGGG)₂ in3 different buffer salt concentrations.

FIG. 11 is a plot showing the change in fluorescence of d(GGGGCCCC)₂,d(CCCCGGGG)₂, d(GGGGCGGGG)₂, d(TGGGCGGGA)₂, and d(TGGGCGGGG)₂ afteraddition of 1r_(dd) neomycin-thiazole orange and thiazole orangecontrol.

FIG. 12 shows the binding of F-neo (42a) to E. coli, Human,Mitochondria, and Mutant Mitochondria A-site oligonucleotide.

FIG. 13 shows a competition dialysis of various nucleic acids using theF-neo (42a) probe.

FIG. 14 shows the relative displacement of TO-neo (36a) from DNA with aminor groove binder versus a major groove binder.

FIG. 15 shows the relative displacement of F-neo (42a) from DNA with aminor groove binder versus a major groove binder.

FIG. 16 shows the displacement of TO-neo (36a) from quadruplex DNA byneomycin-anthraquinone (67).

Like reference numbers and designations in the various drawings indicatelike elements.

DETAILED DESCRIPTION

The present disclosure now will be described more fully hereinafter withreference to specific exemplary embodiments. Indeed, the presentdisclosure can be embodied in many different forms and should not beconstrued as limited to the embodiments set forth herein.

Nucleic acids are well known drug targets. (Dervan, 2001) (Autexier,1999; Ecker & Griffey, 1999; Hurley et al., 2000; Mergny & Helene,1998b; Oganesian & Bryan, 2007; Vicens & Westhof, 2003; Winters, 2000;Zaman, Michiels, & van Boeckel, 2003). Numerous antibacterial andanticancer drugs target nucleic acids such as DNA and RNA. (De et al.,2008; Mergny & Helene, 1998a; Shaw & Arya, 2008; Tor, 2003; Vicens &Westhof, 2003) A rapid way to facilitate nucleic acid target drugdiscovery is the development of methods and probes for high throughputscreening for small molecules that bind nucleic acids such as DNA andRNA. While there are a number of planar molecules that intercalatebetween DNA and RNA base pairs, drugs that bind to the grooves of DNAand RNA structures are expected to be more selective due to thedifferences in groove sizes and widths of different nucleic acids. (Xi,Davis, Ranjan, Xue, Hyde-Volpe, & Arya, 2011a) (Hamilton & Arya, 2012)Therefore it is essential to have such groove binders as lead moleculesfor development of probes that can facilitate the discovery of moreselective and higher affinity molecules.

Aminoglycosides are well known ribosome binding antibacterial drugs.(Arya, 2005; Arya, 2007; Willis & Arya, 2006b) However recent work byArya has shown that a number of aminoglycosides bind nucleic acidgrooves that include a variety of nucleic acid structures such as DNAduplex, (Hamilton & Arya, 2012; Kumar, Xue, & Arya, 2011; Willis & Arya,2010) triplex, (Arya, 2011; Xi, Kumar, Dosen-Micovic, & Arya, 2010; Xueet al., 2010) quadruplex, (Ranjan, Andreasen, Kumar, Hyde-Volpe, & Arya,2010; Xue, Ranjan, & Arya, 2011) DNA. RNA hybrid duplex and triplex,(Arya, Jr., & Charles, 2001; Shaw & Arya, 2008; Shaw, Xi, & Arya, 2008)poly A (Xi, Gray, Kumar, & Arya, 2009) in addition to RNA targets suchas rRNA, HIV TAR RNA, etc. (Arya, Shaw, & Xi, 2007; Charles, Xi, & Arya,2007)

Herein, nucleic acid probes, such as aminoglycoside based fluorescentprobes (Scheme 1), are disclosed.

These disclosed probes can be used for discovery of numerous nucleicacid binding drugs. Nucleic acid targeted screening has mostly relied onthe labeling of the nucleic acid target by a fluorophore. (Karn &Prescott, 2003; Knowles, Karn, Murchie, & Lentzen, 2001) This labelingcan affect the conformation/structure of the target nucleic acid.Attempts to develop small molecule (e.g. aminoglycoside) based probeshave shown some ability to allow screening against a few nucleic acidtargets such as RNA and the ribosome. (Hamasaki & Rando, 1998; Tok, Cho,& Rando, 1999)(Ma et al., 2005; Rando & Wang, 1996) However, even inthese limited applications, the chemistry employed in these applicationsto conjugate the fluorescent probe required the use of amino groups onthe aminoglycosides. This poses significant problems because these aminogroups are inherently required for binding to nucleic acid targets.(Tok, Cho, & Rando, 1999). (Ma et al., 2005; Rando & Wang, 1996).

Amino groups are critical in aminoglycoside binding to nucleic acids.Structural (NMR and X-ray) data has clearly shown the critical roleprotonated amino groups play in aminoglycoside binding (Lynch andPuglisi 2001b; Lynch and Puglisi 2001a; Blanchard, et al. 1998)(Vicensand Westhof 2001; Hermann and Westhof 1998; François, et al). Evidencefor the role of amino groups in binding to negatively charged nucleicacids also comes from the fact that substitution of an amino group(neomycin vs. paromomycin) leads to order(s) of magnitude differences inaffinity (Arya 2011; Xi, et al. 2010; Arya, et al. 2003; Arya, et al.2001). ITC and NMR studies have clearly shown that protonation ofaminoglycoside amino groups is critical upon binding to negativelycharged RNA and DNA structures (Xi, et al. 2010; Barbieri and Pilch2006; Kaul, et al. 2003; Kaul and Pilch 2002).

Additionally, the lack of selectivity of reacting different amines tofluorophore nucleophiles in these embodiments has produced low yields.Attempts were made to better control the selectivity offunctionalization of the aminoglycosides by using a metal basedapproach. (Ghoshal M. & Salamone, 2010). This embodiment however alsoused the amino groups on aminoglycosides required for binding to nucleicacid targets.

Disclosed herein are nucleic acid probes, such as fluorescentaminoglycoside molecules, where fluorescent molecules (e.g. fluorescein,pyrene, methidium, thiazole orange etc) are covalently attached tomonomeric aminoglycosides or dimeric aminoglycosides via selectiveconversion of OH groups on aminoglycosides to reactive groups such asamine, carboxylic acid, isothiocyanate, azide, or alkyne. Thesefluorescent probes bind to a number of nucleic acids such as DNA and RNAand can be displaced by small molecules. The probes are shown here tofunction in a high throughput format for a number of nucleic acidtargets with a Z′>0.5.

The identification of drugs that bind specifically in the major grooveis an important aspect of drug development. The major groove of nucleicacids contains more information for sequence recognition than that ofthe minor groove due to the greater variability of facial functionalgroups available in the grooves. In order to increase the specificity ofnucleic acid binding molecules, it is important to identify leadcompounds that bind to the major groove. The ability of the neomycinbased fluorescent probes to discriminate between molecules that interactin the major minor groove demonstrates that the probes will be useful inthe screening for drugs that bind specifically to the major groove.

In addition to the screening of small molecules that bind to the majorgroove, a logical extension of the use of these fluorescent probes is inthe investigation of nucleic acid binding proteins or othermacromolecules. Similar to the competitive binding assays for the smallmolecules, the displacement of the probes by transcription factors,ribosomal proteins, or other nucleic acid binding proteins wouldidentify proteins that bind within the maj or groove of nucleic acids.Incubation of a nucleic acid binding protein with the nucleic acid probewould result in a change in the fluorescent signal, compared to thenucleic acid probe alone, only if the nucleic acid probe was displacedthrough interactions within the major groove.

Additionally, provided is the combining of a probe based competitionassay with mutated or truncated proteins to identify the domains andresidues that interact with major groove of the nucleic acids. Theinteractions of residues of a protein with the major groove aretypically key elements of a protein's recognition and affinity withnucleic acids. Current methods of measuring the effect of mutations to aprotein's binding affinity, such as filter binding and mobility shiftassays, are slow, requiring multiple steps, and in many cases requireradio-labeled nucleic acid, and only give information on the effects ofthe mutation on binding affinity. Random or directed mutations ofsuspected interacting residues could be quickly screened for theirinteractions within the major groove by comparing the change in thefluorescent signal (i.e., the displacement of the probe describedherein) as a function of the mutation introduced. The probe-based assayis fast, uses a single step following the purification of the protein ina standard lab environment, and is capable of screening the effects ofmultiple mutations in a high throughput format.

The disclosed assays can be performed in a variety of ways, with avariety of steps. For example, some of the steps that can be present inthe assays for inhibitors, such as inhibitors of DNA-proteininteractions using, for example, a neomycin-thiazole orange (TO-neo)conjugate can include: Setting the fluorescence value of the nucleicacid probe, such as TO-neo alone to zero and setting the value of thenucleic acid probe, such as TO-neo, bound to the nucleic acid target toone.

The assays can be performed at different stoichiometric ratios ofnucleic acid and nucleic acid probe, such as a 1:1, 1:2, 1:5, 5:1, or2:1 ratio of the nucleic acid to nucleic acid probe.

Often there is some type of displacement step, where the nucleic acidbound nucleic acid probe (such as TO-neo, or any fluorescent probe asdisclosed herein) is displaced from the nucleic acid, by the normalnucleic acid binding partner, such as a known drug or a known nucleicacid binding protein, such as a DNA binding protein, or even an unknownDNA binding molecule, such as a protein. These displacement steps can beperformed at a variety of molar stoichiometries, of known nucleic acidbinding molecule to the nucleic acid probe:nucleic acid complex, such asa 1:1, 1:2, 1:5, 5:1, or 2:1 ratio. This step can be performed byincubating the nucleic acid probe:nucleic acid complex with the knownDNA binding protein or ligand/drug for example.

As discussed herein, the fluorescent emission of the nucleic acid probeswill change based on binding or non-binding to DNA, and so binding ofthe nucleic acid probe to the nucleic acid, or displacement of thenucleic acid probe from the nucleic acid, or new binding of the nucleicacid probe to the nucleic acid can be done using standard fluorescentmeasurement devices and techniques. For example, fluorescentmeasurements can and have been taken using a plate reader in a 96 wellGreiner black plate.

The emission of fluorescence can be measured at appropriate wavelengthfor the given nucleic acid probe. For example, for TO-neo, the emissionis measured at wavelength 535 nm, using an excitation wavelength of 485nm, averaging the results from multiple measurements.

As there are a number of binding steps in the disclosed assays, such asnucleic acid probe binding nucleic acid, displacement binding steps, andso forth, a variety of buffer conditions can be used for these bindingsteps. For example, most nucleic acid binding proteins bind undersimilar buffer conditions as those shown here for TO-neo binding (10 mMhepes (7.0), 50 mM NaCl, and 0.4 mM EDTA) and would be adaptable tospecific assay conditions.

As the displacement step displaces the nucleic acid probe, there is achange in fluorescence emission. For example, when displacement of theTO-neo by the nucleic acid binding protein or ligand/drug occurs, thisresults in a decrease in fluorescence, if the protein or ligand/drugbinding occurs in the maj or groove, as a function of affinity of theprotein or ligand/drug compared to TO-neo affinity. A detailed discussedis disclosed by Watkins et al. in Ananlytical Biochemistry 434 (2013)300-307 entitled “A fluorescence-based screen for ribosome bindingantibiotics”, which is incorporated herein by reference in its entirety.

An additional aspect to the disclosed assays can be to look at unknownligands for DNA binding, i.e. molecules which may or may not haveaffinity for the major groove or DNA, or some other form of nucleicacid, such as A-form. These unknown ligands can be used in adisplacement step, and their relative abilities to displace the nucleicacid probe, such as TO-neo, can be determined. For example, whenperformed on major groove nucleic acid, ligands that displace TO-neofrom the DNA would be expected to compete with the major groove bindingprotein and lead to identification of inhibitors of DNA-proteininteractions.

As discussed herein, the ability to bind a major groove, or a minorgroove of DNA or RNA or the A-form or B form helix, or even quaternarycomplexes, depends on the aminoglycoside, whether the aminoglycoside isa homodimerized to another aminoglycoside or even another type ofmolecule, such as planar molecules, for example Hoechst 33258. Thedisclosed compounds and compositions, along with the disclosedchemistries are able to make any combination of a fluorescent molecule,a linker, and an aminoglycoside, in either monomeric, homo or heterodimeric form, or derivatized form, with a non-aminoglycoside molecule,such as Hoechst 33258.

Compositions

The disclosed compositions provide nucleic acid probes which comprise afluorophore moiety, a linker moiety, and a Nucleic acid binding moietyillustrated in FIG. 1. In certain embodiments, the fluorophore moleculeis attached to the Nucleic acid binding moiety through the linker suchthat the primary amine groups of the Nucleic acid binding moiety remainfree. In certain embodiments, the primary amine groups of the Nucleicacid binding moiety remain free because the linker is attached to theNucleic acid binding moiety via, for example, the hydroxyl groupsfunctionalized to yield ester, amide, and triazole linkages. The linkagecan be any functional group that that would not use the amino groups ofthe aminoglycoside for linkage. Disclosed are linkages with anaminoglycoside where the linkage occurs through the hydroxyl group of anaminoglycoside. Thus, after attachment of the linker to the Nucleic acidbinding moiety, the primary, secondary, or guanidino amino groups of theNucleic acid binding moiety remain as primary, secondary, or guanidinoamino groups. The nucleic acid binding moieties can include themolecules disclosed herein, including aminoglycosides, homo andheterodimer of aminoglycosides, as well as other nucleic acid bindingmolecules, such as bisbenzamide dyes.

Nucleic Acid Binding Moieties

The nucleic acid binding moieties as disclosed herein, contain at leastone aminosugar, aminoglycoside, or aminoalcohol moiety. An aminosugar isany molecule that has a sugar that has an amino group in it. Anaminoglycoside is any molecule having aminosugars in glycosidic linkage.An aminoalcohol is any molecule that has an amino and a hydroxyl. Thedisclosed nucleic acid binding molecules can be aminoglycosidesconjugated to another aminoglycoside, either the same or different, oraminoglycosides conjugated to other molecules having affinity fornucleic acid, such as planar molecules, for example Hoechst 33258.Examples of nucleic binding moieties can be found in Scheme 2.

Aminoglycosides

PCT/US2006/029675 by Dev P. Arya filed on Jul. 31, 2006 is hereinincorporated by reference in its entirety, but at least for materialrelated to aminoglycosides, nucleic acids, and conjugates of these, aswell as structural information of nucleic acids.

Aminoglycoside antibiotics are bactericidal drugs that have been at theforefront of antimicrobial therapy for almost five decades. The pastdecade (1990-2000) saw a resurgence in aminoglycoside-based drugdevelopment as their chemistry/mechanism of action became betterunderstood. This work, however, had almost exclusively focused ontargeting RNA.

Aminoglycosides are a group of antibiotics that are effective againstcertain types of bacteria. Those which are derived from Streptomycesspecies are named with the suffix-mycin, while those which are derivedfrom micromonospora are named with the suffix-micin. The aminoglycosidesare polar-cations which consist of two or more amino sugars joined in aglycosidic linkage to a hexose nucleus, which is usually in a centralposition. (Chow C S, et al (1997) Chem Rev 97:1489). Though they exhibita narrow toxic/therapeutic ratio, their broad antimicrobial spectrum,rapid bactericidal action, and ability to act synergistically with otherdrags makes them highly effective in the treatment of nosocomial(hospital acquired) infections (Kotra L P, et al (2000) J Urol163:1076). They are clinically useful in the treatment of urinary tractinfections (Santucci R, et al (2000) J Urol 163:1076), lower respiratoryinfections, bacteremias, and other superinfections by resistantorganisms (Forge A, et al (2000) Audio Neurootol 5:3). Their greatestpotential has been in combination drug regimens for the treatment ofinfections that are difficult to cure with single agents and for use inpatients who are allergic to other classes of drugs (Gerding D (2000)Infect Control Hosp Epidemiol 21: S12). Aminoglycosides contain a uniquepolyamine/carbohydrate structure, and have attracted considerableattention because of their specific interactions with RNA (Kaul M, et al(2003) J Mol Biol 326:1373). The bactericidal action of aminoglycosidesis attributed to the irreversible inhibition of protein synthesisfollowing their binding to the 30S subunit of the bacterial ribosome andthus interfering with the mRNA translation process. The miscoding causesmembrane damage, which eventually disrupts the cell integrity, leadingto bacterial cell death (Moazed D, et al (1987) Nature 327:389; PurohitP, et al (1994) Nature 370:659; Recht M I, et al D (1996) J Mol Biol262:421; Miyaguchi H, et al (1996) Nucleic Acids Res 24:3700).

Aminoglycosides include: amikacin, apramycin, arbekacin, bambermycins,butirosin, dibekacin, dibekacin, dihydrostreptomycin, fortimicin,geneticin, gentamicins (e.g., gentamicin A, Cl, CIa, C2 and D),isepamicin, kanamycins (e.g. kanamycin A, B, and C), lividomycin,micronomicin, neamine, neomycins (e.g. neomycin B and C), netilmicin,paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin,streptonicozid, tobramycin, trospectomycin, and viomycin, analogs andderivatives thereof. The free bases, as well as pharmaceuticallyacceptable acid addition salts of these aminoglycoside antibiotics, canbe employed.

Neomycin

In 1995, Mei and co-workers discovered that aminoglycoside antibioticswere able to inhibit Tat peptide binding to the TAR RNA (Mei 1995). Theydiscovered the IC₅₀ values for neomycin, streptomycin, and gentamicin tobe 0.92±0.09 μM, 9.5±0.8 μM, and 45±4 μM, respectively. Their study alsodetermined that the aminoglycosides were bound to the duplex region ofthe RNA, directly below the bulge used for identification by Tat, andthat neomycin B was able to form higher order complexes with the TAR.Further studies on the interactions of neomycin with TAR have since beencompleted, with up to three binding sites identified (Krebs, Ludwig etal. 2003), suggesting that dimeric and trimeric aminoglycosides couldprovide better specificity to TAR. From further studies (CDspectroscopy), it appears that the binding by neomycin induces aconformational change in the RNA, which is different from the usualarchitecture that Tat recognizes, acting as a noncompetitive inhibitorof the Tat-TAR interaction and increasing the rate constant (k_(off))for the dissociation of the peptide (Wang, Huber et al. 1998). Wang etal. also determined that the aminoglycoside binds TAR in the minorgroove, opposite to the major groove binding normally seen. Recently NMRwas used to examine the structural changes that neomycin induces in theTAR RNA; it was found that the neamine core is covered with the bulge,thereby reducing the volume of the major groove in which Tat is normallybound (Faber, Sticht et al. 2000).

The conjugation of fluorescein with neomycin (F-neo, 42a, Scheme 1)allows the molecule to detect the specific binding of the probe to theRNA grooves, DNA major grooves, quadruplex and triplex grooves. Thebinding affinity of the F-neo (42a) also varies slightly with thesequence of the nucleic acid strands and small differences in sequencescan be determined by differences in the fluorescent signal.

The conjugated molecule of fluorescein with the neomycin dimer (79)allows the probe to be directed toward long sequences of DNA (10-14 basepairs). As with the monomer form, F-neodimer 79 allows the detection ofDNA sequences by the change in fluorescence by binding in the majorgroove of DNA.

Major Groove Binders

A major groove binder is a composition or compound which can bind themajor groove of duplex nucleic acid. It is understood that there areB-major groove binders which bind B-form duplex and A-major groovebinders which bind A-form duplex. It is understood that the disclosedprobe can be either B-major groove binders, A-major groove binders ormajor groove binders for DNA conformations between A and B. The majorgroove binders disclosed are exemplary only.

Minor Groove Binders

A minor groove binder is a composition or compound which can bind theminor groove of duplex DNA. It is understood that there are B-minorgroove binders which bind the minor groove of B-form duplex and A-minorgroove binders which bind the minor groove of A-form duplex. It isunderstood that the disclosed probe can have either be A-minor groovebinders or B-minor groove binders conjugated to it. The minor groovebinders disclosed below are exemplary only.

Minor groove recognition relies on van der Waals' contacts, hydrogenbonds, Coulombic attraction and intrinsic properties of the DNA such asflexibility, hydration and electrostatic potential. Successful minorgroove binding ligands typically consist of heterocyclic units such aspyrrole or imidazole groups linked by amides. The flexibility of thesingle bonds between the heterocyclic groups and the amide linkages iscrucial to successful minor groove recognition since the ligand is ableto adopt a twist that matches the helical winding of the DNA, therebypermitting the ligand to maintain contact with the DNA over the foillength of its recognition site. Two thoroughly studied minor groovebinders (MGBs) are Hoechst 33258 (Hoechst) and DAPI, which bindpreferentially at AT-rich regions of B-DNA. Also disclosed are minorgroove binders, such as polyamides, that preferentially bind GC-richregions.

Aminoglycoside Homo and Hetero Dimers

A variety of dimers of aminoglycosides can be conjugated together withinthe disclosed compounds and compositions. For example, certain types ofdimers with certain types of linkages are disclosed in US PatentApplication Publication No. 2011/0046982A1 (herein disclosed andincorporated by reference in its entirety, and at least for dimers andconjugates of aminoglycosides and their synthesis). Other dimers andconjugate of aminoglycosides can be found in WO/2007/016455 (hereindisclosed and incorporated by reference in its entirety, and at leastfor dimers and conjugates of aminoglycosides and their synthesis).

Molecules Having an Affinity for Nucleic Acid

A variety of molecules having an affinity for nucleic acid can beconjugated to one or more aminoglycosides. The bisbenzimide dyes—Hoechst33258, Hoechst 33342 and Hoechst 34580 are cell membrane-permeate, minorgroove-binding DNA stains that fluoresce bright blue upon binding toDNA. Hoechst 33342 has slightly higher membrane permeability thanHoechst 33258, but both dyes are quite soluble in water (up to 2%solutions can be prepared) and relatively nontoxic. Hoechst 34580 hassomewhat longer-wavelength spectra than the other Hoechst dyes whenbound to nucleic acids. These Hoechst dyes, which can be excited withthe UV spectral lines of the argon-ion laser and by most conventionalfluorescence excitation sources, exhibit relatively large Stokes shifts(spectra) (excitation/emission maxima−350/460 nm), making them suitablefor multicolor labeling experiments. The Hoechst 33258 and Hoechst 33342dyes have complex, pH-dependent spectra when not bound to nucleic acids,with a much higher fluorescence quantum yield at pH 5 than at pH 8.Their fluorescence is also enhanced by surfactants such as sodiumdodecyl sulfate (SDS). These dyes appear to show a wide spectrum ofsequence-dependent DNA affinities and bind with sufficient strength topoly(d(A-T)) sequences that they can displace several known DNAintercalators. They also exhibit multiple binding modes and distinctfluorescence emission spectra that are dependent on dye:base pairratios. Hoechst dyes are used in many cellular applications, includingcell-cycle and apoptosis studies and they are common nuclear counterstains. Hoechst 33258, which is selectively toxic to malaria parasites,is also useful for flow-cytometric screening of blood samples formalaria parasites and for assessing their susceptibility to drugs;however, some of the SYTO dyes (cyanine derivatives) disclosed hereinare likely to provide superior performance in these assays.

The Hoechst 33258 and Hoechst 33342 dyes are available as solids (H1398,H1399), as guaranteed high-purity solids (FluoroPure Grade; H21491,H21492) and, for ease of handling, as 10 mg/mL aqueous solutions (H3569,H3570). The Hoechst 34580 dye is available as a solid (H21486).

Others

It has been previously shown that distamycin A binds to the minor grooveof B-form dsDNA (Zimmer C. and Wahnert, U. (1986) Prog. Biophys. Mol.Biol., 47, 31-112). Distamycin A has been shown to preferably bind toDNA duplex tracts containing a 5 bp A-T tract (Kopka M. L., Yoon, C.,Goodsell, D., Pjura, P. and Dickerson, R. E. (1985) Proc. Nat. Acad.Sci. USA, 82, 1376-1380). Netropsin, on the other hand, preferentiallybinds to a DNA duplex tract containing a 4 bp A-T tract (Kopka M. L.,Yoon, C., Goodsell, D., Pjura, P. and Dickerson, R. E. (1985) Proc. Nat.Acad. Sci. USA, 82, 1376-1380).

Among minor groove binders, the N-methylpyrrole carboxamide-containingantibiotics netropsin and distamycin bound to DNA with very pronouncedAT specificity, as expected. More interestingly the dye Hoechst 33258,berenil and a thiazole-containing lexitropsin elicited negative reduceddichroism in the presence of GC-rich DNA which is totally inconsistentwith a groove binding process. These three drugs share with DAPI theproperty of intercalating at GC-rich sites and binding to the minorgroove of DNA at other sites. (Bailly, C, et al. 1992. Drug-DNAsequence-dependent interactions analysed by electric linear dichroism.Journal of Molecular Recognition. 5:4 (155-171).

4-[(3-Methyl-6-(benzothiazol-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-pyridiniumiodide (BEBO) is an asymmetric monovalent cyanine dye that binds in theminor groove of double-stranded (ds)DNA. (Bengtsson, M, et al. A newminor groove binding asymmetric cyanine reporter dye for real-time PCR.Nucleic Acids Res. 2003 Apr. 15; 31(8): e45). Similarly to that of DAPIand Hoechst, the binding of BEBO to poly(dG-dC)₂ is dominated byintercalation, and BEBO has a distinct preference for poly(dA-dT)₂compared to poly(dG-dC)₂.

As judged from the linear and circular dichroism studies, thebenzoxazole derivative BOXTO(4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinoliniumchloride) exhibited straightforward minor groove binding both topoly(dA-dT)₂ and calf thymus DNA (ctDNA), whereas the benzothiazolederivative BEBO showed a more heterogenous binding to the latter DNA,also with a greater tendency for aggregation. (Karlsson, H J, et al.Groove-binding unsymmetrical cyanine dyes for staining of DNA: synthesesand characterization of the DNA-binding. Nucleic Acids Res. 2003 Nov. 1;31(21): 6227-6234).

Linker Moieties

The linker can comprise a backbone of less than 50 atoms, 40 atoms, 30atoms, 20 atoms, and/or alone or in any combination with any otherlimitation or characteristic disclosed herein. Exemplary linkers areshown in Scheme 3.

In certain embodiments the linker can be

In certain embodiments the linker can be-(L₁)_(n)-(L₂)_(m)-(L₃)_(o)-(L₄)_(p)-(L₅)_(q)-(L₆)_(r)-(L₇)_(s)-(L₅)_(t)-(L₉)_(u)-,wherein n, m, o, p, q, r, s, t, u are independently 0 or 1, wherein(L₁), (L₂), (L₃), (L₄), (L₅), (L₆), (L₇), (L₈), and (L₉) areindependently O, N, S, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkoxy, aryl,heteroaryl, heterocyclyl,

and/or alone or in any combination with any other limitation orcharacteristic disclosed herein.

In certain embodiments the linker can be -(L₁)_(v)-, wherein v isindependently 1-20, wherein each (L₁) is independently O, N, S, C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkoxy, aryl, heteroaryl, heterocyclyl,

wherein each (L₁) can be the same or different, and/or alone or in anycombination with any other limitation or characteristic disclosedherein, and/or alone or in any combination with any other limitation orcharacteristic disclosed herein

In certain embodiments the linker can also be O, N, S, C₁-C₈ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkoxy,

and/or alone or in any combination with any other limitation orcharacteristic disclosed herein.

Fluorescence and Fluorescent Moieties

Disclosed are fluorescent moieties which can be attached to thedisclosed compounds. Examples of fluorescent moieties can be found inScheme 4.

The term fluorescent as used herein is defined as a molecule havingluminescence that is caused by the absorption of radiation at onewavelength followed by nearly immediate reradiation usually at adifferent wavelength and that ceases almost at once when the incidentradiation stops, as understood in the art. The term fluorescent labeledmolecule as used herein is defined as a fluorescent labeled molecule ora molecule containing a fluorophore moiety. The term fluorophore moietyas used herein refers to a moiety that has fluorescent properties.Illustrative fluorophore moieties for the present invention includedansyl, 4-(Diethylamino)azobenzene-4′-sulfonyl, fluoresceinisothiocyanate (FITC), 5,6-carboxymethyl fluorescein, Texas red,nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,rhodamine, amino-methyl coumarin (AMCA), Eosin, Erythrosin, BODIPY®,Cascade Blue®, Oregon Green®, pyrene, lissamine, xanthenes, acridines,oxazines, phycoerythrin, macrocyclic chelates of lanthanide ions such asquantum Dye™, fluorescent energy transfer dyes, such as thiazoleorange-ethidium heterodimer, and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5and Cy7; as well as additional examples such as 3-Hydroxypyrene5,8,10-Tri Sulfonic acid, 5-Hydroxy Tryptamine (5-HT), Acid Fuchsin,Alizarin Complexon, Alizarin Red, Allophycocyanin, Aminocoumarin,Anthroyl Stearate, Astrazon Brilliant Red 4G, Astrazon Orange R,Astrazon Red 6B, Astrazon Yellow 7 GLL, Atabrine, Auramine,Aurophosphine, Aurophosphine G, BAO 9 (Bisaminophenyloxadiazole), BCECF(2′,7′-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein), BerberineSulphate, Bisbenzamide, Blancophor FFG Solution, Blancophor SV, BodipyF1, Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, CalcofluorRW Solution, Calcofluor White, Calcophor White ABT Solution, CalcophorWhite Standard Solution, Carbostyryl, Cascade Yellow, Catecholamine,Chinacrine, Coriphosphine O, Coumarin-Phalloidin, CY3.1 8, CY5.1 8, CY7,Dans (1-Dimethyl Amino Naphaline 5 Sulphonic Acid), Dansa (DiaminoNaphtyl Sulphonic Acid), Dansyl NH—CH₃, Diamino Phenyl Oxydiazole (DAO),Dimethylamino-5-Sulphonic acid, Dipyrrometheneboron Difluoride, DiphenylBrilliant Flavine 7GFF, Dopamine, Erythrosin ITC, Euchrysin, FIF(Formaldehyde Induced Fluorescence), Flazo Orange, Fluo 3,Fluorescamine, Fura-2, Genacryl Brilliant Red B, Genacryl BrilliantYellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF, Gloxalic Acid,Granular Blue, Haematoporphyrin, Indo-1, Intrawhite Cf Liquid, LeucophorPAF, Leucophor SF, Leucophor WS, Lissamine Rhodamine B200 (RD200),Lucifer Yellow CH, Lucifer Yellow VS, Magdala Red, Marina Blue, MaxilonBrilliant Flavin 10 GFF, Maxilon Brilliant Flavin 8 GFF, MPS (MethylGreen Pyronine Stilbene), Mithramycin, NBD Amine, Nitrobenzoxadidole,Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan BrilliantFlavin E8G, Oxadiazole, Pacific Blue, Pararosaniline (Feulgen), PhorwiteAR Solution, Phorwite BKL, Phorwite Rev, Phorwite RPA, Phosphine 3R,Phthalocyanine, Phycoerythrin R, Polyazaindacene Pontochrome Blue Black,Porphyrin, Primuline, Procion Yellow, Pyronine, Pyronine B, PyrozalBrilliant Flavin 7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5GLD, Rhodamine 6G, Rhodamine B, Rhodamine B 200, Rhodamine B Extra,Rhodamine BB, Rhodamine BG, Rhodamine WT, Serotonin, Sevron BrilliantRed 2B, Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange,Sevron Yellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonicacid), Stilbene, Snarf 1, sulpho Rhodamine B Can C, Sulpho Rhodamine GExtra, Tetracycline, Thiazine Red R, Thioflavin S, Thioflavin TCN,Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, True Blue,Ultralite, Uranine B, Uvitex SFC, Xylene Orange, and XRITC.

Probe Equilibrium

The following equilibrium is used to help explain the probe function:

where FHNeo represents the protonated form of F-neo. F(−)Neo, uponbinding to nucleic acids such as DNA or RNA, is destabilized by thenucleic acid backbone negative potential, shifting the equilibriumtowards FH, making F-neo a weaker acid, raising the pKa, which is whatwe observe upon binding to the A-site. For the A-site targeted assay, wecalculated the Z′ factor (Z=1-3(2.7+2.8)/(414-267)=0.89. A value of Z′=1is ideal, Z′ between 0.5 and 1 is considered excellent.

Synthesis of Disclosed Compounds and Compositions

The compounds and compositions disclosed herein and the compounds andcompositions necessary to perform the disclosed methods can be madeusing any method known to those of skill in the art for that particularreagent or compound unless otherwise specifically noted. It isunderstood that the disclosed reactions are representative, and can beused to make the disclosed compounds and compositions, but variousappropriate substitutions can be made and are understood.

Functionalization of Neomycin

Neomycin, like other aminoglycosides, such as neamine and kanamycin, canbe functionalized in several ways to afford functional groups that serveas the centers of electrophilic/nucleophilic reactivity as shown inScheme 5.

In addition to this, they can also be useful towards copper catalyzedclick chemistry reactions. From commercially available neomycin BSulfate, the first step of reaction is protection of all (such as six)amino groups (scheme 6a) using tertiary butyl carbonyl (Boc) groups,which lead to the formation of Boc protected neomycin 1. The protectionof amino groups by Boc renders the solubility properties of neomycinfrom being water soluble to complete insolubility in water. This alsoallows much easier purification of the desired product from byproductsformed in the reaction. Neomycin has four rings, three of them (rings I,II and IV) contain amino groups on them while ring III contains a loneprimary hydroxyl present in the molecule at the 5-position, in additionto a secondary hydroxyl group at 3-position in the ring (scheme 6a). Theprimary hydroxyl can then be selectively reacted with a bulky leavinggroup such as triisopropyl benzene sulfonyl chloride (TPS-C1) whichleads to the formation of compound 2. Conversion of the primary hydroxylgroup on ring III to a good leaving group TPS facilitates smoothnucleophilic reactions at this position. The TPS functional group can bedisplaced in two ways. First, 2-aminoethanethiol can be deprotonated inthe presence of base to displace the TPS group present in 2, therebygiving an amine terminated Boc protected neomycin 3 which can beconjugated directly. The TPS group can also be displaced by reaction ofsodium azide in the presence of a polar protic solvent to afford anazide terminated Boc protected neomycin 5 which can be used straight forclick chemistry. Compound 5 can be reduced in the presence of aheterogeneous catalyst to its corresponding amine 6 in quantitativeyields. Thus, neomycin can be derivatized into amine functionalities intwo ways which give us differing atom spacing at the 5″-end on ring III.The two —NH₂ terminated neomycin derivatives (3 or 6) can be convertedto their corresponding isothiocyanates in a single step by reaction withTCDP (Charles, Xue, & Arya, 2002). The isothiocyanate functionality nowserves as an electrophilic center for reactions with nucleophilicligands. Neomycin amine 6 can then also be used to prepare a carboxylterminated neomycin derivative 8 by reaction with succinic anhydride inthe presence of DMF. Neomycin amine 6 can also be reacted with propargylchloroformate in the presence of base to give a short alkyne terminatedneomycin derivative 7 (Kumar, 2011) that reacts with azide partners in afacile way using copper catalyzed click chemistry. (Kolb, Finn, &Sharpless, 2001; Rostovtsev, Green, Fokin, & Sharpless, 2002) Compound 2can also be reacted with 2-mercaptoethyl ether to give thiol endedneomycin derivative 10 which can be used in thiol maleimide couplingreactions. The primary hydroxyl group in 1 can be reacted underMitsunobu conditions to form an aldehyde ended neomycin derivative 9that can be used for condensation with amines. Alkyne terminatedneomycin derivatives (with a triazole ring on its linker) can also beprepared by reaction with excess bisalkynes to give alkyne terminatedneomycin derivatives with varying atoms spacing. A generalized scheme ofthe reaction is outlined in scheme 6b.

Scheme 6a. Scheme showing the modification of neomycin to acid, alkyne,amine, thiol, aldehyde or isothiocyanate functionalized aminosugar.Reagents and Conditions: (a) (Boc)₂O, Et₃N, H₂O, 75° C., 18 h, 75% (b)TPS-Cl, pyridine, rt, 3 days, 61% (c) NaN₃, DMF:H₂O (10:1), 80° C., 12h, 90% (d) Na(metal), ethanol(dry), HSCH₂CH₂NH₂.HCl, rt, 10 h, 70% (e)TCDP, DCM, rt, 24 h, 50% (f) Pd—C, H2, rt, 12 h, qaunt. (g) Succinicanhydride, rt, overnight, DMF (h)Propargyl chloroformate, pyridine, rt,overnight, 60% (i) 4-hydroxy benzaldehyde, TPP, DIAD, Toluene (k)2-mercaptoethyl ether, Cs₂CO₃, rt, 12 h, 92%.

Functionalization of Neamine (Riguet et al., 2005; Riguet, Désiré,Bailly, & Décout, 2004)

Functionalization of Neamine requires protection of both amino andhydroxyl groups as shown in scheme 7. In the first step, neamine isreacted with trityl chloride in the presence of base to afford tritylprotected amines 12. Reaction of the tetratritylated derivative ofneamine with three equivalents of 4-methoxy benzyl chloride leads toboth bis and tri benzylated products which can be purified using columnchromatography to give 13. The 5-hydroxyl group can then be deprotonatedin the presence of sodium hydride and then be reacted with excess alkylbis bromides to give a bromo ended derivative 14a. The bromo group canbe converted to azido functionality 14 by reaction with sodium azide inDMF. The azide functionality in 14 can be reduced to its correspondingamine 15 by reaction with PPh₃ in THF. The amine functionality in 15 canthen be converted to various other functional groups. The amine can bereacted with TCDP to give its corresponding isothiocyanate 17. The aminecan also be reacted with succinic anhydride in the presence of DMF togive carboxyl terminated neamine derivative 16. It can also be reactedwith propargyl chloroformate in the presence of base to give alkyneterminated neamine derivative 18. The hydroxyl group in 13 can bereacted under Mitsunobu conditions to form an aldehyde ended neaminederivative 19 that can be used for condensation with amines. Compound14a can also be reacted with 2-mercaptoethyl ether to give thiol endedneamine derivative 20 which can be used in thiol maleimide couplingreactions.

Functionalization of Kanamycin (Wang, 1998)(Charles, Xue, & Arya, 2002)

Functionalization of Kanamycin 21 starts with the Cbz protection of thefive amino groups in the presence of a base (scheme 8). In the nextstep, the lone primary hydroxyl group present on the molecule isconverted to a good leaving group in the form of triisopropyl benzenesulfonyl (TPS) derivative 22. In the next step, the Cbz groups can beremoved by hydrogenolysis and the amino groups can be Boc protected in aone pot reaction. The TPS group can then be displaced by 2-amino ethanethiolate to give an amine terminated Kanamycin 23, which can be used forcoupling reactions with isothiocyanates or ligands with free carboxylicacid groups. The amine 23 can also be converted to its correspondingisothiocyanate 24 by reaction with TCDP. Compound 22 can also beconverted to an azido derivative 25 by a combination of hydrogenolysisand Boc protection of amino groups followed by the displacement of the-TPS group by sodium azide in DMF. The azide 25 can be reduced to ashorter amine 26 by palladium catalyzed hydrogenation, which can then beused to make either a carboxyl terminated derivative 27 by reaction withsuccinic anhydride or an alkyne terminated derivative by reaction withpropargyl chloroformate 28. The amine group in 26 can be reacted with4-(2-isothiocyanatoethoxy)benzaldehyde to form an aldehyde endedkanamycin derivative 29 that can be used for condensation with amines.The TPS leaving group in 22 can also be reacted with 2-mercaptoethylether to give thiol ended kanamycin derivative 30 which can be used inthiol maleimide coupling reactions. The complete route to the synthesesto these derivatives is outlined in scheme 8.

Compositions and Methods

Disclosed are nucleic acid probes comprising a fluorescent moiety, alinker moiety, and a nucleic acid binding moiety, wherein thefluorescent molecule is covalently attached to the linker moiety and thelinker moiety is covalently attached to the nucleic acid binding moietythrough a carbon or oxygen or sulfur atom of the nucleic acid bindingmoiety, examples show in Scheme 9. The linker is not bound to thenucleic acid binding moiety through an amino group of the nucleic acidbinding moiety.

Disclosed are nucleic acid probes, wherein the fluorescent moiety isfluorophore, for example shown in Scheme 4. The linker moiety comprisesa backbone of less than 50 atoms, wherein the linker moiety comprises abackbone of less than 40 atoms, wherein the linker moiety comprises abackbone of less than 30 atoms, wherein the linker moiety comprises abackbone of less than 20 atoms, wherein the linker moiety comprises alinker shown in Scheme 3, wherein the nucleic acid binding moietycomprises a compound shown in scheme 2, alone and/or in any combinationwith the compounds, compositions, and methods disclosed herein.

Disclosed are kits comprising a nucleic acid probe disclosed herein.

Disclosed are methods comprising incubating a nucleic acid with anucleic acid probe disclosed herein, wherein the probe comprises afluorophore, wherein fluoresence intensity of the fluorophore changes ina manner proportional to how tightly the probe is binding the nucleicacid.

In some embodiments, the fluorophore is fluorescein, which upon bindingcauses its fluorescence to decrease. In some embodiments, thefluorophore is thiozaole-orange, which upon binding causes thefluorescence to increase, alone and/or in any combination with thecompounds, compositions, and methods disclosed herein.

Disclosed are methods comprising 1) incubating an oligonucleotide with anucleic acid probe disclosed herein under conditions where the probedisclosed herein can bind the oligonucleotide to form a complex, 2)measuring a fluorescence emission intensity of the complex, 3)incubating a compound with the complex to form a incubation mixture, 4)measuring a fluorescence emission intensity on the incubation mixture,5) comparing the fluorescence emission intensity of the complex and theincubation mixture to determine the binding affinity of the compoundrelative to the probe.

Disclosed are methods comprising, incubating a substrate with a nucleicacid probe disclosed herein, wherein the nucleic acid probe comprises afluorophore, the fluoresence intensity of which changes in a mannerproportional to how tightly the probe is binding the substrate, whereinthe substrate is RNA, single stranded DNA, A-dsDNA, B-dsDNA, or fourstranded DNA.

Pharmaceutical Carriers/Delivery of Pharmaceutical Products

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include primers to perform theamplification reactions discussed in certain embodiments of the methods,as well as the buffers and enzymes required to use the primers asintended.

Compositions with Similar Functions

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures which can perform the same function which arerelated to the disclosed structures, and that these structures willultimately achieve the same result.

Process Claims for Making the Compositions

Disclosed are processes for making the compositions as well as making,the intermediates leading to the compositions. There are a variety ofmethods that can be used for making these compositions, such assynthetic chemical methods and standard molecular biology methods. It isunderstood that the methods of making these and the other disclosedcompositions are specifically disclosed.

Methods of Using the Compositions as Research Tools

The disclosed compositions can be used in a variety of ways as researchtools. For example, the disclosed compositions, can be used to study theinteractions between aminoglycosides and nucleic acids, by for example,identifying new molecules that bind the nucleic acids.

The compositions can be used for example as tools within combinatorialchemistry protocols or other screening protocols to isolate moleculesthat possess desired functional properties related to their nucleic acidbinding.

The disclosed compositions can be used as discussed herein as, eitherreagents in micro arrays or as reagents to probe or analyze existingmicroarrays. The compositions can also be used in any known method ofscreening assays, related to chip/micro arrays.

DEFINITIONS

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.As used herein, the term “activity” refers to a biological activity. Theterm “cell” as used herein also refers to individual cells, cell lines,or cultures derived from such cells. A “culture” refers to a compositioncomprising isolated cells of the same or a different type. The termco-culture is used to designate when more than one type of cell arecultured together in the same dish with either full or partial contactwith each other. Compounds and compositions have their standard meaningin the art. It is understood that wherever, a particular designation,such as a molecule, substance, marker, cell, or reagent compositionscomprising, consisting of, and consisting essentially of thesedesignations are disclosed. Thus, where the particular designationmarker is used, it is understood that also disclosed would becompositions comprising that marker, consisting of that marker, orconsisting essentially of that marker. Where appropriate wherever aparticular designation is made, it is understood that the compound ofthat designation is also disclosed. For example, if particularbiological material, such as aminoglycoside, is disclosed aminoglycosidein its compound form is also disclosed. Disclosed are materials,compounds, compositions, and components that can be used for, can beused in conjunction with, can be used in preparation for, or areproducts of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if an inhibitor is disclosed and discussed and anumber of modifications that can be made to a number of R groups arediscussed, each and every combination and permutation of the inhibitorand the modifications to its R group that are possible are specificallycontemplated unless specifically indicated to the contrary. Thus, if aclass of substituents A, B, and C are disclosed as well as a class ofsubstituents D, E, and F and an example of a combination molecule, A-Dis disclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, in this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.

The term “aldehyde” as used herein is represented by the formula —C(O)H.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon moiety. “Unbranched” or “Branched” alkyls comprise anon-cyclic, saturated, straight or branched chain hydrocarbon moietyhaving from 1 to 24 carbons, 1 to 12, carbons, 1 to 6 carbons, or 1 to 4carbon atoms. Examples of such alkyl radicals include methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, n-propyl, iso-propyl,butyl, n-butyl, sec-butyl, t-butyl, amyl, t-amyl, n-pentyl and the like.Lower alkyls comprise a noncyclic, saturated, straight or branched chainhydrocarbon residue having from 1 to 4 carbon atoms, i.e., C1-C4 alkyl.Moreover, the term “alkyl” as used throughout the specification andclaims is intended to include both “unsubstituted alkyls” and“substituted alkyls”, the later denotes an alkyl radical analogous tothe above definition that is further substituted with one, two, or moreadditional organic or inorganic substituent groups. Suitable substituentgroups include but are not limited to hydroxyl, cycloalkyl, amino,mono-substituted amino, di-substituted amino, unsubstituted orsubstituted amido, carbonyl, halogen, sulfhydryl, sulfonyl, sulfonato,sulfamoyl, sulfonamide, azido, acyloxy, nitro, cyano, carboxy,carboalkoxy, alkylcarboxamido, substituted alkylcarboxamido,dialkylcarboxamido, substituted dialkylcarboxamido, alkylsulfonyl,alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy,haloalkoxy, heteroaryl, substituted heteroaryl, aryl or substitutedaryl. It will be understood by those skilled in the art that an “alkoxy”can be a substituted of a carbonyl substituted “alkyl” forming an ester.When more than one substituent group is present then they can be thesame or different. The organic substituent moieties can comprise from 1to 12 carbon atoms or from 1 to 6 carbon atoms, or from 1 to 4 carbonatoms. It will be understood by those skilled in the art that themoieties substituted on the “alkyl” chain can themselves be substituted,as described above, if appropriate.

The term “alkenyl” as used herein is an alkyl residue as defined abovethat also comprises at least one carbon-carbon double bond in thebackbone of the hydrocarbon chain. Examples include but are not limitedto vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl,4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexanyl, 2-heptenyl,3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl and the like. The term“alkenyl” includes dienes and trienes of straight and branch chains.

The term “alkynyl” as used herein is an alkyl residue as defined abovethat comprises at least one carbon-carbon triple bond in the backbone ofthe hydrocarbon chain. Examples include but are not limited ethynyl,1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl,2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,4-hexynyl, 5-hexynyl and the like. The term “alkynyl” includes di- andtri-ynes.

The term “alkoxy” as used herein is an alkyl residue, as defined above,bonded directly to an oxygen atom, which is then bonded to anothermoiety. Examples include methoxy, ethoxy, n-propoxy, iso-propoxy,n-butoxy, t-butoxy, iso-butoxy and the like

The term “aryl” as used herein is a ring radical containing 6 to 18carbons, or preferably 6 to 12 carbons, comprising at least one aromaticresidue therein. Examples of such aryl radicals include phenyl,naphthyl, and ischroman radicals. Moreover, the term “aryl” as usedthroughout the specification and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the later denotes anaryl ring radical as defined above that is substituted with one or more,preferably 1, 2, or 3 organic or inorganic substituent groups, whichinclude but are not limited to a halogen, alkyl, alkenyl, alkynyl,hydroxyl, cycloalkyl, amino, mono-substituted amino, di-substitutedamino, unsubstituted or substituted amido, carbonyl, halogen,sulfhydryl, sulfonyl, sulfonato, sulfamoyl, sulfonamide, azido acyloxy,nitro, cyano, carboxy, carboalkoxy, alkylcarboxamido, substitutedalkylcarboxamido, dialkylcarboxamido, substituted dialkylcarboxamido,alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy,substituted alkoxy or haloalkoxy, aryl, substituted aryl, heteroaryl,heterocyclic ring, ring wherein the terms are defined herein. Theorganic substituent groups can comprise from 1 to 12 carbon atoms, orfrom 1 to 6 carbon atoms, or from 1 to 4 carbon atoms. It will beunderstood by those skilled in the art that the moieties substituted onthe “aryl” can themselves be substituted, as described above, ifappropriate.

The term “acyl” as used herein is a R—C(O)— residue having an R groupcontaining 1 to 8 carbons. Examples include but are not limited toformyl, acetyl, propionyl, butanoyl, iso-butanoyl, pentanoyl, hexanoyl,heptanoyl, benzoyl and the like, and natural or un-natural amino acids.

As used herein, the term “azide”, “azido” and their variants refer toany moiety or compound comprising the monovalent group —N₃ or themonovalent ion —N₃.

The term “acyloxy” as used herein is an acyl radical as defined abovedirectly attached to an oxygen to form an R—C(O)O— residue. Examplesinclude but are not limited to acetyloxy, propionyloxy, butanoyloxy,iso-butanoyloxy, benzoyloxy and the like.

The term backbone atom when used herein with respect to a linker refersto an atom in the shortest direct path of covalent bonding between thetwo chief moieties that are linked by the linker.

The term “carbonate group” as used herein is represented by the formula—OC(O)OR, where R can be hydrogen, an alkyl, alkenyl, alkynyl, aryl,aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl groupdescribed above.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH.

The term “carbonyl group” as used herein is represented by the formulaC═O.

The term “cycloalkyl” as used herein is a saturated hydrocarbonstructure wherein the structure is closed to form at least one ring.Cycloalkyls typically comprise a cyclic radical containing 3 to 8 ringcarbons, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclopenyl,cyclohexyl, cycloheptyl and the like. Cycloalkyl radicals can bemulticyclic and can contain a total of 3 to 18 carbons, or preferably 4to 12 carbons, or 5 to 8 carbons. Examples of multicyclic cycloalkylsinclude decahydronapthyl, adamantyl, and like radicals.

Moreover, the term “cycloalkyl” as used throughout the specification andclaims is intended to include both “unsubstituted cycloalkyls” and“substituted cycloalkyls”, the later denotes an cycloalkyl radicalanalogous to the above definition that is further substituted with one,two, or more additional organic or inorganic substituent groups that caninclude but are not limited to hydroxyl, cycloalkyl, amino,mono-substituted amino, di-substituted amino, unsubstituted orsubstituted amido, carbonyl, halogen, sulfhydryl, sulfonyl, sulfonato,sulfamoyl, sulfonamide, azido, acyloxy, nitro, cyano, carboxy,carboalkoxy, alkylcarboxamido, substituted alkylcarboxamido,dialkylcarboxamido, substituted dialkylcarboxamido, alkylsulfonyl,alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy,haloalkoxy, heteroaryl, substituted heteroaryl, aryl or substitutedaryl. When the cycloalkyl is substituted with more than one substituentgroup, they can be the same or different. The organic substituent groupscan comprise from 1 to 12 carbon atoms, or from 1 to 6 carbon atoms, orfrom 1 to 4 carbon atoms.

The term “cycloalkenyl” as used herein is a cycloalkyl radical asdefined above that comprises at least one carbon-carbon double bond.Examples include but are not limited to cyclopropenyl, 1-cyclobutenyl,2-cyclobutenyl, 1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl,1-cyclohexyl, 2-cyclohexyl, 3-cyclohexyl and the like.

The term “di-substituted amino” as used herein is a moiety comprising anitrogen atom substituted with two organic radicals that can be the sameor different, which can be selected from but are not limited to aryl,substituted aryl, alkyl, substituted alkyl or arylalkyl, wherein theterms have the same definitions found throughout. Some examples includedimethylamino, methylethylamino, diethylamino and the like.

The term “ether” as used herein is represented by the formula AOA¹,where A and A¹ can be, independently, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “ester” as used herein is represented by the formula —C(O)OA,where A can be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl,heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, orheterocycloalkenyl group described above.

The term “haloalkyl” as used herein is an alkyl residue as definedabove, substituted with one or more halogens, preferably fluorine, suchas a trifluoromethyl, pentafluoroethyl and the like.

The term “haloalkoxy” as used herein a haloalkyl residue as definedabove that is directly attached to an oxygen to form trifluoromethoxy,pentafluoroethoxy and the like.

The term “halo” or “halogen” refers to a fluoro, chloro, bromo or iodogroup.

The term “heteroaryl” as used herein is an aryl ring radical as definedabove, wherein at least one of the ring carbons, or preferably 1, 2, or3 carbons of the aryl aromatic ring has been replaced with a heteroatom,which include but are not limited to nitrogen, oxygen, and sulfur atoms.Examples of heteroaryl residues include pyridyl, bipyridyl, furanyl, andthiofuranyl residues. Substituted “heteroaryl” residues can have one ormore organic or inorganic substituent groups, or preferably 1, 2, or 3such groups, as referred to herein-above for aryl groups, bound to thecarbon atoms of the heteroaromatic rings. The organic substituent groupscan comprise from 1 to 12 carbon atoms, or from 1 to 6 carbon atoms, orfrom 1 to 4 carbon atoms.

The term “heterocyclyl” or “heterocyclic group” as used herein is anon-aromatic mono- or multi ring radical structure having 3 to 16members, preferably 4 to 10 members, in which at least one ringstructure include 1 to 4 heteroatoms (e.g. O, N, S, P, and the like).Heterocyclyl groups include, for example, pyrrolidine, oxolane,thiolane, imidazole, oxazole, piperidine, piperizine, morpholine,lactones, lactams, such as azetidiones, and pyrrolidiones, sultams,sultones, and the like. Moreover, the term “heterocyclyl” as usedthroughout the specification and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the later denotes anaryl ring radical as defined above that is substituted with one or more,preferably 1, 2, or 3 organic or inorganic substituent groups, whichinclude but are not limited to a halogen, alkyl, alkenyl, alkynyl,hydroxyl, cycloalkyl, amino, mono-substituted amino, di-substitutedamino, unsubstituted or substituted amido, carbonyl, halogen,sulfhydryl, sulfonyl, sulfonato, sulfamoyl, sulfonamide, azido acyloxy,nitro, cyano, carboxy, carboalkoxy, alkylcarboxamido, substitutedalkylcarboxamido, dialkylcarboxamido, substituted dialkylcarboxamido,alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy,substituted alkoxy or haloalkoxy, aryl, substituted aryl, heteroaryl,heterocyclic ring, ring wherein the terms are defined herein. Theorganic substituent groups can comprise from 1 to 12 carbon atoms orfrom 1 to 6 carbon atoms, or from 1 to 4 carbon atoms. It will beunderstood by those skilled in the art that the moieties substituted onthe “heterocyclyl” can themselves be substituted, as described above, ifappropriate.

The term “keto group” as used herein is represented by the formula—C(O)R, where R is an alkyl, alkenyl, alkynyl, aryl, aralkyl,cycloalkyl, halogenated alkyl, or heterocycloalkyl group describedabove.

As used herein, the terms “linked”, “operably linked” and “operablybound” and variants thereof mean, for purposes of the specification andclaims, to refer to fusion, bond, adherence or association of sufficientstability to withstand conditions encountered in single moleculeapplications and/or the methods and systems disclosed herein, between acombination of different molecules such as, but not limited to: betweena detectable label and nucleotide, between a detectable label and alinker, between a nucleotide and a linker, between a protein and afunctionalized nanocrystal; between a linker and a protein; and thelike. For example, in a labeled polymerase, the label is operably linkedto the polymerase in such a way that the resultant labeled polymerasecan readily participate in a polymerization reaction. See, for example,Hermanson, G., 2008, Bioconjugate Techniques, Second Edition. Suchoperable linkage or binding may comprise any sort of fusion, bond,adherence or association, including, but not limited to, covalent,ionic, hydrogen, hydrophilic, hydrophobic or affinity bonding, affinitybonding, van der Waals forces, mechanical bonding, etc.

The term “linker” and its variants, as used herein, include any compoundor moiety that can act as a molecular bridge that operably links twodifferent molecules. There are many different linkers and typesdisclosed herein, such as those designated with a —B—.

The term “metabolite” refers to active derivatives produced uponintroduction of a compound into a biological milieu, such as a patient.

The term “urethane” as used herein is represented by the formula—OC(O)NRR′, where R and R′ can be, independently, hydrogen, an alkyl,alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, orheterocycloalkyl group described above.

The term “mono-substituted amino” as used herein is a moiety comprisingan NH radical substituted with one organic substituent group, whichinclude but are not limited to alkyls, substituted alkyls, cycloalkyls,aryls, or arylalkyls. Examples of mono-substituted amino groups includemethylamino (—NH—CH₃); ethylamino (—NHCH₂CH₃), hydroxyethylamino(—NH—CH₂CH₂OH), and the like.

A “moiety” is part of a molecule (or compound, or analog, etc). A“functional group” is a specific group of atoms in a molecule. A moietycan be a functional group or can include one or functional groups.

The term “silyl group” as used herein is represented by the formula—SiRR′R″, where R, R′, and R″ can be, independently, hydrogen, an alkyl,alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, alkoxy,or heterocycloalkyl group described above.

When used with respect to pharmaceutical compositions, the term “stable”is generally understood in the art as meaning less than a certainamount, usually 10%, loss of the active ingredient under specifiedstorage conditions for a stated period of time. The time required for acomposition to be considered stable is relative to the use of eachproduct and is dictated by the commercial practicalities of producingthe product, holding it for quality control and inspection, shipping itto a wholesaler or direct to a customer where it is held again instorage before its eventual use. Including a safety factor of a fewmonths time, the minimum product life for pharmaceuticals is usually oneyear and preferably more than 18 months. As used herein, the term“stable” references these market realities and the ability to store andtransport the product at readily attainable environmental conditionssuch as refrigerated conditions, 2° C. to 8° C.

The term “sulfo-oxo group” as used herein is represented by the formulas—S(O)₂R, —OS(O)₂R, or, —OS(O)₂OR, where R can be hydrogen, an alkyl,alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, orheterocycloalkyl group described above.

A “detection agent” or like terms refers to any molecule or moiety whichcan be detected by, such as flourescence, radioactivity,phosphorescence, or the like.

The terms “higher,” “increases,” “elevates,” or “elevation” or variantsof these terms, refer to increases above basal levels, e.g., as comparedto a control. The terms “low,” “lower,” “reduces,” or “reduction” orvariation of these terms, refer to decreases below basal levels, e.g.,as compared to a control. For example, basal levels are normal in vivolevels prior to, or in the absence of, or addition of an agent such asan agonist or antagonist to activity.

By “inhibit” or other forms of inhibit means to hinder or restrain aparticular characteristic. It is understood that this is typically inrelation to some standard or expected value, in other words it isrelative, but that it is not always necessary for the standard orrelative value to be referred to. For example, “inhibitsphosphorylation” means hindering or restraining the amount ofphosphorylation that takes place relative to a standard or a control.

References in the specification and concluding claims to parts byweight, of a particular element or component in a composition orarticle, denotes the weight relationship between the element orcomponent and any other elements or components in the composition orarticle for which a part by weight is expressed. Thus, in a compoundcontaining 2 parts by weight of component X and 5 parts by weightcomponent Y, X and Y are present at a weight ratio of 2:5, and arepresent in such ratio regardless of whether additional components arecontained in the compound.

A weight percent of a component, unless specifically stated to thecontrary, is based on the total weight of the formulation or compositionin which the component is included.

A “labeled RNA binder” or like terms refers to a molecule comprising adetection agent.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

As used herein, the term “pharmacological activity” refers to theinherent physical properties of a peptide or polypeptide. Theseproperties include but are not limited to half-life, solubility, andstability and other pharmacokinetic properties.

“Primers” are a subset of probes which are capable of supporting sometype of enzymatic manipulation and which can hybridize with a targetnucleic acid such that the enzymatic manipulation can occur. A primercan be made from any combination of nucleotides or nucleotidederivatives or analogs available in the art which do not interfere withthe enzymatic manipulation.

“Probes” are molecules capable of interacting with a target nucleicacid, typically in a sequence specific manner, for example throughhybridization. The hybridization of nucleic acids is well understood inthe art and discussed herein. Typically a probe can be made from anycombination of nucleotides or nucleotide derivatives or analogsavailable in the art.

By “prevent” or other forms of prevent means to stop a particularcharacteristic or condition. Prevent does not require comparison to acontrol as it is typically more absolute than, for example, reduce orinhibit. As used herein, something could be reduced but not inhibited orprevented, but something that is reduced could also be inhibited orprevented. It is understood that where reduce, inhibit or prevent areused, unless specifically indicated otherwise, the use of the other twowords is also expressly disclosed. Thus, if inhibits phosphorylation isdisclosed, then reduces and prevents phosphorylation are also disclosed.

The term “pro-drug or prodrug” is intended to encompass compounds which,under physiologic conditions, are converted into therapeutically activeagents. A common method for making a prodrug is to include selectedmoieties which are hydrolyzed under physiologic conditions to reveal thedesired molecule. In other embodiments, the prodrug is converted by anenzymatic activity of the host animal.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data are provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular datum point “10” and a particular datum point 15 aredisclosed, it is understood that greater than, greater than or equal to,less than, less than or equal to, and equal to 10 and 15 are considereddisclosed as well as between 10 and 15. It is also understood that eachunit between two particular units are also disclosed. For example, if 10and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

By “reduce” or other forms of reduce means lowering of an event orcharacteristic. It is understood that this is typically in relation tosome standard or expected value, in other words it is relative, but thatit is not always necessary for the standard or relative value to bereferred to. For example, “reduces phosphorylation” means lowering theamount of phosphorylation that takes place relative to a standard or acontrol.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

As used throughout, by a “subject” is meant an individual. Thus, the“subject” can include, for example, domesticated animals, such as cats,dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.),laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals,non-human mammals, primates, non-human primates, rodents, birds,reptiles, amphibians, fish, and any other animal. The subject can be amammal such as a primate or a human.

“Treating” or “treatment” does not mean a complete cure. It means thatthe symptoms of the underlying disease are reduced, and/or that one ormore of the underlying cellular, physiological, or biochemical causes ormechanisms causing the symptoms are reduced. It is understood thatreduced, as used in this context, means relative to the state of thedisease, including the molecular state of the disease, not just thephysiological state of the disease.

The term “therapeutically effective” means that the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination. Theterm “carrier” means a compound, composition, substance, or structurethat, when in combination with a compound or composition, aids orfacilitates preparation, storage, administration, delivery,effectiveness, selectivity, or any other feature of the compound orcomposition for its intended use or purpose. For example, a carrier canbe selected to minimize any degradation of the active ingredient and tominimize any adverse side effects in the subject.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

Example 1 Development of a Fluorescence-Based Assay for AminoglycosideBinding to Target RNA

A survey of a library of fluorescently-labeled neomycin derivatives wasperformed, and a compound exhibiting decrease in fluorescence intensityupon binding to the bacterial rRNA A-site oligonucleotide wasidentified. This compound F-neo or 42a is a fluorescein conjugatecoupled through the 5″ OH position of the ribose ring of neomycin B (H.Xi, E. Davis, N. Ranjan, L. Xue, D. Hyde-Volpe, D. P. Arya,Thermodynamics of nucleic Acid “shape readout” by an aminosugar,Biochemistry. 50 (2011) 9088-9113). E. coli rRNA Asite oligonucleotide(SEQ ID NO. 1, GGCGUCACACCUUCGGGUGAAGUCGCC) is a synthetic RNA sequence27 bases long designed to mimic the ribosomal A-site. The A-siteoligonucleotide (SEQ ID NO. 1) is titrated into F-neo (42a) to give aconcentration dependent loss of fluorescence of F-neo (42a). Thetitration of Scatchard analysis indicates one binding site of F-neo(42a) per oligonucleotide and a dissociation constant of 2.3×10⁷.Molecular modeling of the interaction of F-neo (42a) with A-siteoligonucleotide suggests that the fluorescein moiety localizes in thegroove in a manner that places the phenol group adjacent to thephosphate backbone.

The effect of bacterial A-site oligonucleotide (SEQ ID No. 1) on thefluorescence emission intensity of a fluorescein-neomycin conjugate(F-neo or 42a) is recorded in FIG. 2. FIG. 2A shows that the emissionspectra of 0.25 mM F-neo excited at 490 nm is line 100 and with theaddition of 0.3 mM bacterial rRNA A-site oligonucleotide (SEQ ID No. 1)it lowered to line 102. FIG. 2B shows the effect of titration offluorescence emission (517 nm) of 0.3 mM F-neo with increasingconcentrations of A-site oligonucleotide (SEQ ID No. 1). Experimentswere performed in 10 mM MOPSO, 0.4 mM EDTA, and 50 mM NaCl at pH 7.0. Asshown in FIG. 2, the addition of a 1:1 stoichiometric ratio of conjugate(F-neo) and A-site oligonucleotide resulted in a 3-fold reduction influorescence emission intensity.

The F-neo (42a) can be competitively displaced from the A-site RNAoligonucleotide (SEQ ID No. 1) by the addition of non-fluorescent RNAbinding drugs. Titrating neomycin into the F-neo (42a):A-site complexincreases the fluorescence in a dose dependent manner that saturates ata 1:1 stoichiometric ratio of drug-to-complex. Additionally, a strongsignal window, measured as an increase in fluorescence is present at a3:1 molar ratio, and a maximum change in fluorescence at approximately10:1 molar ratio. Titrating in neomycin reverses this effect in a dosedependent manner that saturated at a 1:1 stoichiometric ratio ofdrug-to-complex was shown in FIG. 3. Fluorescence emission intensity(517 nm) of 0.3 mM A-site oligonucletide/F-neo complex solutionincreased with increasing concentration of neomycin. Experiments wereperformed in 10 mM MOPSO, 0.4 mM EDTA, and 50 mM NaCl at pH 7.0. Thisprovides an example of how the assay system can function as a powerfulassay for assessing the binding affinity of aminoglycosides and theirderivatives towards RNA targets.

Example 2 Development of a High-Throughput Screening Assay (HTS)

Provided is a detailed example for the development of a HTS by thedisplacement of a fluorescene-neomycin (F-neo (42a)) from rRNA A-siteoligonucleotide (SEQ ID No. 1) using a 96 well plate format and afluorescent plate reader.

The high throughput screening (HTS) determines the binding of drugs tothe ribosomal A-site. This HTS involves the displacement of afluorescene-neomycin (F-neo (42a)) by potential RNA binding molecules ina 96 well plate format using a fluorescent plate reader. Initial studiesof this system have demonstrated that the binding of F-neo (42a) toA-site RNA results in the quenching of the fluorophore, and thedisplacement of F-neo (42a) by a competitive binding molecule ismeasured by an increase in fluorescence.

F-neo based RNA binding assays have been performed using a fluorescencespectrophotometer with a 96-well plate reader and results recorded inFIG. 4. FIG. 4A shows the duplicate measurements of the fluorescenceemission intensity (517 nm) of 0.3 mM F-neo alone; 0.30 mM F-neo and 0.3mM A-site oligonucleotide; and 0.30 mM F-neo, 0.3 mM A-siteoligonucleotide and 1.0 mM neomycin. Duplicate F-neo samples in the leftpanel show low variation in fluorescence and responded to the additionof A-site with 2-fold reduction in fluorescence intensity. Moreover, theaddition of neomycin to samples of F-neo/A-site oligonucleotide complexrestored the intensity to F-neo only levels. FIG. 4B shows duplicatemeasurements of samples with 0.30 mM F-neo, 0.3 mM A-siteoligonucleotide and 1.0 mM of one of neomycin, paromomycin, orribostamycin. Sample volume was 200 mL and solutions contained 10 mMMOPSO, 0.4 mM EDTA, and 50 mM NaCl at pH 7.0. Fo is defined as thebaseline fluorescence intensity of the RNA/F-neo complex and F is theintensity with added drug. The difference between the fluorescenceintensity baseline of F-neo/A-site complex and complex with added drug(F-Fo) indicates the extent of the competition of drug for the bindingsite, this allows the relative binding affinities of aminoglycosides tobe established. It has been reported that the relative bindingaffinities for neomycin, paromomycin, and ribostamycin with bacterialA-site oligos is neomycin >paromomycin >ribostamycin (Kaul, 2002). Asseen in FIG. 4 right panel, the 96-well format of this competition assaywas effective at assessing the relative binding affinity of theseaminoglycosides. FIG. 4C shows the screening of aminoglycoside librarywith 10 Scans per Well. Aminoglycosides 0.3 μM neomycin, paromomycin,ribostamycin, neamine, gentamycin, hygromycin, and streptomycin wereadded to 0.1. M E. coli A-site/F-neo (42a) complex. Each result wastaken from the average of ten experiments ran on a 96 well Greiner blackplate. The emission was measured at wavelength 535 nm, using anexcitation wavelength of 485 nm using a Tecan Genios Pro plate reader.All experiments were performed in 10 mM hepes (7.0), 50 mM NaCl, and 0.4mM EDTA. F-Fo is calculated by subtracting the fluorescence of theexperiment with the aminoglycoside present from the fluorescence of theA-site/F-neo (42a) complex in the absence of the aminoglycoside. Usingthe difference between the fluorescence intensity baseline of F-neo(42a)/A-site complex and complex with added drug (F-Fo) indicates theextent of the competition of drug for the binding site. This analysisallows the relative binding affinities of aminoglycosides to beestablished. These results further show that a F-neo (42a) based assaycan be used for efficient high throughput screening of small moleculelibraries such as aminoglycoside libraries for drug candidates.

The quality of the assay was determined by the calculation of aZ′-factor using equation 1 for the displacement of F-neo (42a) from theE. coli A-site by neomycin.

Z′-factor=1−3×(σ_(p)+σ_(n))/|μ_(p)−μ_(n)|  Eq. (1)

The final assay results were obtained using the average (n) and standarddeviation (on) from 48 wells of 0.1 μM F-neo (42a)/E. coli ribosomalA-site as the negative control and from the average (μp) and standarddeviation (σ_(p)) 48 wells of 0.1 μM F-neo (42a)/E. coli ribosomalA-site mixed with 0.3 μM neomycin as the positive control. The Z′-factorwas calculated from the HTS with 0.3 μM neomycin to 0.1 μM E coliA-site/F-neo (42a) complex using 100 reads/well using a Tecan Genios Proplate reader. Each bar represents an independent experiment ran on thedesignated day. Each Z′ was calculated using the average and standarddeviation of 48 wells on a 96 well black plate. The emission wasmeasured at wavelength 535 nm, using an excitation wavelength of 485 nm.All experiments were performed in 10 mM hepes (7.0), 50 mM NaCl, and 0.4mM EDTA. Three independent experiments performed on three consecutivedays resulted in Z′-factor of 0.81, 0.80, and 0.87, indicated the highdegree of reproducibility of the assay. FIG. 5 shows the results ofthese experiments. The accepted scale of the Z′-factor is >0.5 isexcellent, 0<Z′<0.5 is marginal, and Z′<0 is unacceptable. Our resultsindicate that the assay is suitable for the detection drugs that bind tothe ribosomal A-site of E. coli by the displacement of the F-neo (42a)probe in a high throughput format.

Example 3 The pH Sensitivity of the Probe and Binding Site ElectrostaticPotential

The mechanism for change in fluorescence of F-Neo upon binding to A-siterRNA oligonucleotide (SEQ ID No. 1) using UV absorption spectroscopy wasinvestigated. Effect of pH on the absorbance spectra of 3.0 mMfluorescein-neomycin in 20 mM MOPSO, 0.4 mM EDTA, 50 mM NaCl at theindicated pH values were studied. FIG. 6A shows the absorbance spectraof 3.0 mM fluorescein-neomycin at pH 7.10 (106), pH 5.95 (108) and pH2.5 (110). FIG. 6B shows the absorbance at 490 nm offluorescein-neomycin as function of pH.

The effect of A-site rRNA oligonucleotide (SEQ ID No. 1) on theabsorbance spectra of fluorescein-neomycin at pH 7.1 was recorded inFIG. 7. The absorbance spectra of 3.0 mM fluorescein-neomycin in 20 mMMOPSO, 0.4 mM EDTA, 50 mM NaCl at pH 7.1 with the addition of 0 mM(112), 1.1 mM (114), 2.2 mM (116) or 3.3 mM (118) of A-siteoligonucleotide (SEQ ID NO. 1), respectively is shown in FIG. 7A.Absorbance at 490 nm of fluorescein-neomycin:A-site complex as functionof pH is show in FIG. 7B, with 3.0 mM fluorescein-neomycin complexedwith 3.3 mM A-site oligonucleotide (SEQ ID No. 1) in 20 mM MOPSO, 0.4 mMEDTA, 50 mM NaCl at the indicated pH values.

With increasing pH, fluorescein-neomycin undergoes a transition betweenmono-anion and di-anion species. Fluorescein-neomycin concentration was10 μM in 100 mM NaCl, 10 mM SC, 0.5 mM EDTA. As indicated by itsabsorbance spectra in FIG. 8A, the F-neo transitions from a mono-anionto a di-anion with increasing pH. FIG. 8B compares the absorbancespectra between the mono-anion and di-anion species of fluoresceinneomycin. FIG. 8C shows absorbance maximum plotted as a function of pHwith the resulting inflection point representing the fluoresceinphenolic pKa. The absorbance peak of about 490 nm in FIGS. 8A and 8B isconsistent with the phenolate species of fluorescein. The pKa for theequilibrium of the phenol and phenolate forms of F-neo (42a) wasmeasured by evaluating the absorbance spectrum as a function of pH andshown to be 6.84. This is very similar to the pka of 6.7 for reportedfor free fluorescein (martin and lindqvist, 1975) indicating theneomycin moiety does not alter this pKa appreciably. When monitoring theabsorption spectrum in the presence of the A-site RNA (SEQ ID No. 1), areduction in A₄₉₀ consistent with the change in pKa was observed. ThisA₄₉₀ reduction is consistent with a shift in equilibrium towards theprotonated non-fluorescent form of fluorescein-neomycin upon binding toA-site. This titration curve suggests that the binding of A-sitestabilizes the non-fluorescent protonated form of fluorescein-neomycin,due to an upward shift of the pKa of approximately one pH unit. Thisshift in pKa causes a change in the fluorescence intensity of F-neo(42a). The titration of F-neo (42a) with A-site oligonucleotide at pH7.1 resulted in a decrease in the fluorescence observed at 490 nm,consistent with a shift for the highly fluorescent phenolate form to thenon-fluorescent phenol form of the dye.

The following equilibrium can be used to explain the probe function:

FHNeo+Base=F(−)Neo+BH ⁺  Eq. (2)

where FHNeo represents the protonated form of F-neo (42a). Upon bindingto A-site RNA, F(−)Neo is destabilized by the negative potential of theRNA backbone, shifting the equilibrium towards FHNeo. Thus the bindingof F-neo (42a) to RNA raises the pKa, making F-neo (42a) a weaker acid.

Since it has been shown that a shift in phenolic pKa is directlyproportional to the electrostatic potential (Ψ) at the position occupiedby the fluorescein moiety (Friedrich, K., et al., (1988) Eur. J Biochem.173, 227-231.) it can be concluded that the shift in pKa between freeFluorescein-neomycin and Fluorescein-neomycin bound to DNA's majorgroove reveals the electrostatic potential of the major groove,neomycin's binding site. The local electrostatic potential (Ψ) in themajor groove is given by the following Equation 3.

Ψ=−[R•T• ln 10/(N _(A) •e)]ΔpKa  Eq. (3)

Where ΔpKa is the difference between the free Fluorescein-neomycin andthe Fluorescein-neomycin in the presence of the electrostaticenvironment, R is the universal gas constant, T is the absolutetemperature in kelvin, N_(A) is the Avogadro constant, and e is theelementary charge. R=8.312 J mol−1K−1, T=298.2K, N_(A)=6.023×10²³ ande=1.602×10⁻¹⁹; Ψ is expressed in mV. Thus a shift of +1 in the value ofthe pKa implies an electrostatic potential of −58.2 mV. The pH-dependentabsorbance spectra of F-neo (42a)/A-site oligonucleotide complexesindicated that RNA binding causes a shift apparent pKa of F-neo (42a) to7.7. Based on this Δpka of 0.86, the electrostatic potential of themicroenvironment for the fluorescein moiety in the complex is determinedto be −50.9 mV.

Determination of the pKa was done using chemometric analysis of theabsorption profiles at various pH values of the covalently attachedfluorescein moiety (Kubista, M., et al., (1993) Anal. Chem. 65, 994-998,Kubista, M., et al., (1995) Anal. Chim. Acta. 302, 121-125.2, 3). The pHdependent absorption profile of free fluorescein-neomycin was firstanalyzed to determine its pKa. This was then repeated with one molarequivalent of d(GGGGCCCC)₂ and d(CCCCGGGG)₂ and the effect of thesenucleic acids resulted in a pKa shift of the fluorescein-neomycin asrecorded in FIG. 9. The electrostatic potential (P) calculated from theshift in pKa, and the ITC derived binding constant (K). Electrostaticpotential experiments performed in 100 mM NaCl, 10 mM SC, 0.5 mM EDTA at25° C. ITC experiments performed in 50 mM KCl, 10 mM SC, 0.5 mM EDTA (pH6.0) at 25° C. As shown in FIG. 9, the shift in absorbance maximumplotted as a function of pH for fluorescein-neomycin with one molarequivalent d(CCCCGGGG)₂ (122), and d(GGGGCCCC)₂ (124) compared to freefluorescein-neomycin (120) resulting in the shift in pKa.Fluorescein-neomycin concentration was 10 μM. Table 1 represents the pKavalues determined from the inflection point of the pH dependentabsorbance scans of free fluorescein-neomycin, fluorescein-neomycin withone molar equivalent d(CCCCGGGG)₂ and d(GGGGCCCC)₂. The electrostaticpotential (Y) calculated from the shift in pKa, and the ITC derivedbinding constant (K).

TABLE 1 Oligonucleotide pKa ΔpKa Ψ (mV) K (M⁻¹) × 10⁵ FreeFluorescein-neomycin 6.57 — — — d(CCCCGGGG)₂ 6.83 0.26 −15.13 7.97 ±10.9 d(GGGGCCCC)₂ 7.01 0.44 −25.61 66.3 ± 9.7 

With the change in pKa the electrostatic potential of the neomycinbinding site was calculated using equation (2). The shift in phenolicpKa of fluorescein-neomycin in the presence of d(GGGGCCCC)₂ wasconsiderably greater than the shift seen in the presence ofd(CCCCGGGG)₂. Using the shifts in pKa the electrostatic potentials ofeach respective binding site was determined. The electrostatic potentialof the neomycin binding site of d(GGGGCCCC)₂ was calculated to be −25.61mV and d(CCCCGGGG)₂ was calculated to be −15.13 mV. The substantialdifference in electrostatic potential between the two inverted duplexesd(GGGGCCCC)₂ and d(CCCCGGGG)₂ reflects a difference in electrostaticcomplementarity between the DNA duplexes and neomycin.

Electrostatic potential is a vital force driving the intermolecularassociation and molecular properties of small molecules (Weiner, P. K.,et al., (1982) PNAS. 79, 3754-37584), actions of drug molecules, andanalogs (Weinstein, H., et al., (1975) Mol. Pharmacol. 11, 671-6895),and enzyme catalysis (Warshel, A., (1981)Acc. Chem. Res. 14, 284-2906).Fluorescein has been exploited as a DNA probe to monitor hybridformation in solution (Murakami, A., et al. (1991) Nucl. Acids Res. 19,4097-41027), and with covalent attachment the electrostatic potentialaround the nucleic acid (Sjoback, R., et al., (1998) Biopolymers. 46,445-4538). Here it was shown that a fluorescein-neomycin conjugate canbe used to determine the electrostatic potential of DNA's major groove,the aminosugar binding site. The binding site specific electrostaticpotentials of two inverted DNA duplexes were compared with theITC-derived binding constants. The comparison between electrostaticpotential and ITC-derived binding constants illustrated the influence ofelectrostatic potential on the affinity of neomycin. CD experimentsperformed show the difference in DNA conformations between d(GGGGCCCC)₂and d(CCCCGGGG)₂. Drug-DNA recognition is dependent on the structuralelectrostatic complementarity (Arya, D. P. (2005) The Case for Neomycin,in Topics in Current Chemistry: DNA Binders (J. B. Chaires, and M. J.Waring, Eds.) pp 149-178, Springer Verlag: Heidelburg, 9). Thedifferences in CD spectra provide evidence showing how the difference inelectrostatic potential of the maj or groove binding site is affected byconformational differences.

Example 4 Use of F-Neo as a High Throughput Screening Probe to IdentifyHigh Affinity Major Groove Binding Sequences

It has been shown that the fluorescence of a fluorescein-neomycin isquenched when bound to a nucleic acid target. In continued efforts toexpand the applications of the fluorescein-neomycin conjugate, afluorescence quenching study was performed to determine if the quenchingof fluorescein-neomycin in the presence of DNA is representative of theneomycin affinity. Previous studies using the duplexes d(GGGGCCCC)₂ andd(CCCCGGGG)₂ have shown the binding is lower for d(CCCCGGGG)₂. This hasbeen shown from the ITC derived binding constants, FID studies, andelectrostatic potential studies. With these results thefluorescein-neomycin quenching for d(GGGGCCCC)₂ and d(CCCCGGGG)₂ isshown here, and also salt dependent studies here have shown neomycinbinding decreases with increasing buffer salt concentration. For thisreason the fluorescence quenching of fluorescein-neomycin withd(GGGGCCCC)₂ and d(CCCCGGGG)₂ was performed at three different buffersalt concentrations. If the fluorescein-neomycin quenching is directlyrelated to the affinity for neomycin then d(GGGGCCCC)₂ would quench thefluorescence better than d(CCCCGGGG)₂ and the fluorescein-neomycinquenching for both duplexes would decrease with increasing buffer saltconcentrations, as seen In Table 2. As the salt concentration isincreased from 50 to 150 mM NaCl, AF values for both the DNA sequencesdrop significantly (from 73% to 33% and from 47% to 20%). Results fromthis study show here that the fluorescein-neomycin quenching can beapplied to screening the 256 GC rich hairpins to identify which sequenceneomycin has the highest affinity. Table 2 represents the change influorescence (%) values of fluorescein-neomycin with lrdr d(GGGGCCCC)₂and d(CCCCGGGG)₂ in 3 different buffer salt concentrations and the ratiobetween the change in fluorescence (%) values of F-neo with d(CCCCGGGG)₂compared to d(GGGGCCCC)₂. Fluorescein-neomycin=1 μM, Buffer=10 mM SC,0.5 mM EDTA, and either 50 mM NaCl, 100 mM NaCl, or 150 mM NaCl(pH=6.8). Experiment was performed in triplicate and the results werealso polted in FIG. 10 showing the change in fluorescence (%) offluorescein-neomycin with 1 equivalent d(GGGGCCCC)2 and d(CCCCGGGG)2 in3 different buffer salt concentrations.

TABLE 2 Ratio of ΔF (%) with Buffer Salt ΔF (%) with 1r_(dr) ΔF (%) with1r_(dr) d(CCCCGGGG)₂/ Concentration d(GGGGCCCC)₂ d(CCCCGGGG)₂d(GGGGCCCC)₂  50 mM NaCl 73.97 ± 5.78 47.75 ± 5.60 0.65 100 mM NaCl49.28 ± 1.12 32.18 ± 0.42 0.65 150 mM NaCl 33.88 ± 3.33 20.36 ± 2.760.60

The results from the fluorescein-neomycin quenching experiment show thequenching of fluorescein-neomycin is related to the affinity of the DNAduplex for neomycin. In addition the effect of buffer salt concentrationcontinues to show the fluorescein-neomycin quenching is correlated withthe binding of fluorescein-neomycin which represents the binding forneomycin. Also the difference between the quenching offluorescein-neomycin with d(GGGGCCCC)₂ and d(CCCCGGGG)₂ shows the sameratio of change in the different salt concentrations showing the bindingof both d(GGGGCCCC)₂ and d(CCCCGGGG)₂ are both equally affected by thesalt concentration of the buffer. This method of fluorescein-neomycinquenching can therefore be used for screening nucleic acid sequences ofvarying affinities.

Example 5 High Throughput Sequence Specificity Screening with a ThiazoleOrange-Neomycin Conjugate

Neomycin-thiazole orange was made with 2 different linker lengths TO-neo1 (36b) and TO-neo2 (36a) and the chemical structures shown below.

The change in fluorescence of d(GGGGCCCC)₂, d(CCCCGGGG)₂, d(GGGGCGGGG)₂,d(TGGGCGGGA)₂, and d(TGGGCGGGG)₂ in 50 mM NaCl, 10 mM SC, 0.5 mM EDTA(pH 6.0) after addition of 1r_(dd) neomycin-thiazole orange and thiazoleorange control were recorded in FIGS. 11A and 11B, respectively. Withthe addition of one molar equivalent of five various DNA sequences thefluorescence increase varies based on the affinity of neomycin-thiazoleorange for the DNA duplex. This is indicated by the largest fluorescenceincrease. When a control experiment was performed with thiazole orangealone the results differed. This is to be expected since thiazole orangehas some sequence specificity, although the pattern between the duplexeswas different for the neomycin-thiazole orange. This indicates not onlydoes the fluorescence increase correlated with the binding ofneomycin-thiazole orange but also the sequence specificity forneomycin-thiazole orange is different than thiazole orange alone.

To gain a better understanding of the sequence specificity of neomycinconjugate 36a and 36b, a screening assay was performed with 256 GC richhairpin duplexes. After showing neomycin-thiazole orange binding can bedetermined using five different DNA duplexes, the high throughputapplications were demonstrated using 256 DNA hairpins with varying GCcontent. The assay using 256 DNA duplexes allows for determining thehighest affinity sequence without the added variables of a pre-boundfluorescent marker such as ethidium bromide. In addition the magnitudeof fluorescence enhancement of neomycin-thiazole orange allows for theassay to easily be performed on a 96 well plate reader.

The results from the 256 DNA hairpins assay show the highly sequencedependent affinity of neomycin-thiazole orange. In addition the assayshows how the neomycin-thiazole orange affinity for a particular duplexin a large library can be identified from a simple screening assay.Since this assay eliminates the need for a pre-bound fluorescent markerthe assay is a simple 2 step process. First the baseline fluorescence ofneomycin-thiazole orange is recorded, then DNA is added and thefluorescence is again recorded, and the assay is complete giving enoughinformation for identifying the highest affinity sequences for furtherstudy. Addition of unlabeled drug to the DNA:TO-Neo2 complex displacesthe TO-Neo from DNA leading to a decrease in fluorescence. The assay cantherefore screen for target specificity as well as screen for drugbinding to a specific target.

Example 6 The High Throughput Screening (HTS) of Mutations in Sequencesof RNA

Disclosed is an HTS showing the difference in the binding affinity offluorescene-neomycin (F-neo (42a)) to various synthetic RNA sequence 27bases long designed to mimic the ribosomal A-site using a 96 well plateformat and a fluorescent plate reader. Differences in binding aredetermined by the differential quenching of F-neo (42a) upon binding tothe RNA.

The affinity of F-neo (42a) shows small but measurable differences formutated RNA sequences. In order to demonstrate the ability to detectthese differences similar ribosomal A-site mimics of E. coli ribosomalA-site (GGCGUCACACCUUCGGGUGAAGUCGCC) (SEQ ID No. 1), human(GGCGUCGCUACUUCGGUAAAAGUCGCC) (SEQ ID NO. 2), mitochondria(GGCGUCACCCCUUCGGGACAAGUCGCC) (SEQ ID NO. 3), and a mutant mitochondria(GGCGUCACCCCUUCGGGGCAAGUCGCC) (SEQ ID NO. 4) ribosomal A-sites(highlighted in bold are bases that each sequence differs from E. colisequence). Following the SOP's established for F-neo (42a) binding to E.coli A-site, each sequence was added at 0.1 μM RNA to 0.1 μM F-neo (42a)using 100 reads/well using a Tecan Genios Pro plate reader in a 96 wellGreiner black plate. The emission was measured at wavelength 535 nm,using an excitation wavelength of 485 nm. All experiments were performedin 10 mM hepes (7.0), 50 mM NaCl, and 0.4 mM EDTA. Ten wells wereaveraged for each sequence and measurable differences were observed inthe quenching of F-neo (42a), with fluorescence for E. coli of 7963(±221), human of 9503 (±208), mitochondria of 11987 (±445), and mutantmitochondria of 8515 (±230).

The results of the A-site screening demonstrated the ability of theassay to detect small mutations in RNA sequences is shown in FIG. 12.The application of the assay can be extended to screen and to detectdrug binding to mutants in other important RNAs such as siRNA, mRNA,miRNA, as well as others, and the scope of the assay is not limited tothe sequences chosen here.

Example 7 Major Groove Binding—High Throughput Screening of GC DNABinding Drugs

The HTS by the displacement of a fluorescene-neomycin (F-neo (42a))bound from a synthetic DNA sequence eight bases long designed to conformto A-form DNA by the synthetic molecule neomycin-anthraquinone (67)using a 96 well plate format and a fluorescent plate reader isdisclosed.

The high throughput screening (HTS) has been designed to determine thebinding of drugs to the target sequence d(G₄C₄). The target is predictedto exist predominately in the A-form conformation of DNA. This HTSinvolves the displacement of a fluorescene-neomycin (F-neo (42a)) by DNAbinding molecules in a 96 well plate format using a fluorescent platereader. Initial studies of this system have demonstrated that thebinding of F-neo to A-form DNA results in the quenching of thefluorophore, and the displacement of F-neo (42a) by a competitivebinding molecule is measured by an increase in fluorescence. The qualityof the assay was determined by the calculation of a Z′-factor usingequation 1 for the displacement of F-neo (42a) from the DNA byneomycin-anthraquinone 67. The final assay results were obtained usingthe average (μ_(n)) and standard deviation (σ_(n)) from 36 wells of 0.1μM F-neo (42a)/DNA d(G₄C₄) as the negative control and from the average(μ_(p)) and standard deviation (σ_(p)) 36 wells of 0.1 μM F-neo(42a)/DNA d(G₄C₄) mixed with 0.3 μM neomycin-anthraquinone 67 as thepositive control. The experiment resulted in Z′-factor of 0.84. Theresults indicate that the assay is suitable for the detection of drugsthat bind to the DNA d(G₄C₄) by the displacement of the F-neo (42a)probe in a high through-put format.

Example 8 Specific GC Rich DNA Binding

The HTS by the displacement of a thiazole-neomycin (TO-neo (36a)) boundfrom a synthetic DNA sequence eight bases long designed to conform toA-form DNA by the synthetic molecule neomycin-anthraquinone 67 using a96 well plate format and a fluorescent plate reader was disclosedherein.

The high throughput screening (HTS) has been designed to determine thebinding of drugs to the target sequence d(G₄C₄). The target is predictedto exist predominately in the A-form conformation of DNA. This HTSinvolves the displacement of a TO-neo (36a) molecule by DNA bindingmolecules in a 96 well plate format using a fluorescent plate reader.Initial studies of this system have demonstrated that the binding ofTO-neo (36a) to GC DNA results in flourescence, and the displacement ofTO-neo (36a) by a competitive binding molecule causes a loss offluorescence. The quality of the assay was determined by the calculationof a Z′-factor using equation 1 for the displacement of TO-neo (36a)from the DNA by a neomycin-anthraquinone 67 conjugate. The final assayresults were obtained using the average (μ_(n)) and standard deviation(σ_(n)) from 24 wells of 1.0 μM TO-neo (36a)/DNA d(G₄C₄) as the negativecontrol and from the average (μ_(p)) and standard deviation (σ_(p)) 24wells of 1.0 μM F-neo (42a)/DNA d(G₄C₄) mixed with 3.0 μMneomycin-anthraquinone 67 as the positive control. The experimentresulted in Z′-factor of 0.83. Our results indicate that the assay issuitable for the detection drugs that bind to the DNA d(G₄C₄) by thedisplacement of the TO-neo (36a) probe in a high through-put format.

Example 9 High Throughput Screening of AT Rich DNA Binding Drugs

The HTS by the displacement of a fluorescene-neomycin dimer (F-neodimer79) bound from a synthetic DNA hairpin d(A₁₂A₅T₁₂) designed to conformto B-form DNA by the synthetic molecule compound 60 using a 96 wellplate format and a fluorescent plate reader is disclosed.

The high throughput screening (HTS) has been designed to determine thebinding of drugs to AT rich sequence d(A₁₂A₅T₁₂). The target ispredicted to exist predominately in the B-form conformation of DNA. ThisHTS involves the displacement of an F-neodimer 79 by DNA bindingmolecules in a 96 well plate format using a fluorescent plate reader.Initial studies of this system have demonstrated that the binding ofF-neodimer 79 to B-form DNA results in the quenching of the fluorophore,and the displacement of 79 by a competitive binding molecule is measuredby an increase in fluorescence. The quality of the assay was determinedby the calculation of a Z′-factor using equation 1 for the displacementof F-neodimer 79 from the DNA by compound 60. The final assay resultswere obtained using the average (μn) and standard deviation (σ_(n)) from36 wells of 0.5 μM F-neodimer 79/d(A₁₂A₅T₁₂) as the negative control andfrom the average (μ_(p)) and standard deviation (σ_(p)) 36 wells of 0.5μM 79/d(A₁₂A₅T₁₂) mixed with 1.5 μM compound DPA 51 (synthesis reportedin Kumar, et. al Biochemistry, 2011) as the positive control. Theexperiment resulted in Z′-factor of 0.54. Our results indicate that theassay is suitable for the detection of drugs that bind to a B-form DNAd(A₁₂A₅T₁₂) by the displacement of the F-neodimer 79 probe in a highthrough-put format.

Example 10 Development of Probes for Detection of ConformationalDifference Nucleic Acid

Disclosed is the use of fluorescent aminosugar probes such as F-neo(42a) to probe a variety of nucleic acid targets. Competition dialysiswas used as a nucleic acid screening technique to illustrate the bindingof F-neo (42a) to different nucleic acid structures.

Competition dialysis studies using a fluorescein-neomycin conjugate(F-neo (42a)). As a control, binding of fluorescein to nucleic acids wasinvestigated in the competition dialysis assay and showed negligiblebinding. Comparative binding of F-neo (42a) to 13 different nucleic acidstructures was examined which included single strand nucleic acids[poly(dT), poly(A), poly(U)], duplexes [poly(dA)•poly(dT),poly(rA)•poly(dT), poly(rA)•poly(rU), poly(dA)•poly(rU),poly(dG)•poly(dC), calf thymus], triplexes [poly(dA)•2poly(dT),poly(rA)•2poly(rU)], i-motif (polydC) and 16S A-site rRNA (SEQ ID No.1). The amount of F-neo (42a) bound to each nucleic acid is shown as abar graph in FIG. 13. Because all nucleic acids are dialyzedsimultaneously in the same ligand solution, the amount of bound F-neo(42a) is directly proportional to its affinity for each nucleic acid.

FIG. 13a,c , e shows competition dialysis results offluorescein-neomycin with various nucleic acids at (a) 100 mM Na⁺ (c),150 mM Na⁺ (e) 200 mM Na⁺. FIG. 13 b,d,f shows competition dialysisresults of fluorescein with various nucleic acids at (b) 100 mM Na⁺ (d)150 mM Na⁺ (f) 200 mM Na⁺. 200 μL of different nucleic acids (7.5 μM permonomeric unit of each polymer) was dialyzed for 72 h with 400 mL 100 nMligand in 6 mM Na₂HPO4, 2 mM NaH2PO4, 1 mM Na₂EDTA, pH 7.0 and variousNa⁺ concentrations as indicated.

F-neo (42a) prefers to bind to 16S A-site rRNA yielding a bound ligandconcentration of 85 nM in 100 mM Na⁺, 50 nM in 150 mM Na⁺, and 15 nM in200 mM Na⁺ is shown in FIG. 15. Binding to the RNA triplexpoly(rA)•2poly(rU) is comparable to A-site rRNA under low saltconditions, with 60 nM, 20 nM, and 5 nM bound F-neo (42a) in 100, 150,and 200 mM Na⁺ respectively. F-neo (42a) also shows significant bindingto RNA duplex poly(rA)•poly(rU), DNA triplex poly(dA)•2poly(dT), andhybrid duplex poly(dA)•poly(rU) with approximately 10-20 nM bound drugunder these salt conditions. However, F-neo (42a) exhibits moderate tovery weak binding with the DNA duplex poly(dA)•poly(dT), hybrid duplexpoly(rA)•poly(dT), calf thymus DNA, i-motif DNA, and all single-strandednucleic acids studied here.

The binding of F-neo (42a) to nucleic acids is affected by the saltconcentration. As Na⁺ concentration increases from 100 to 200 mM, theamount of bound neomycin decreases approximately 4-5 times for eachnucleic acid structure. Additionally, the counterintuitive andsurprising absence of F-neo (42a) binding to poly(dA)•2poly(dT) under100 mM Na⁺ can be explained by the non-formation of the DNA triplexunder this salt concentration at ambient temperatures (˜22° C.). As saltconcentration is raised to 150 mM Na⁺, triplex formation is favored andligand binding is observed.

Competition dialysis reveals that F-neo (42a) binds the natural target16S A-site rRNA and also shows comparable binding to RNA triplex.Analysis of the nucleic acid structures that are favored by neomycinsuggests that they all display features characteristic of A-formconformation. The hybrid duplex, especially poly(dA)•poly(rU), exhibitsintermediate conformation between the A and B forms with a higherpropensity for A-form. The low bound drug observed for poly(rA)•poly(dT)can be attributed to the fact that this hybrid can exist in the B-form.In addition, GC-rich sequences are well known to adopt A-likeconformation in aqueous solution. Highly ordered nucleic acidsstructures such as G-quadruplex and triplex also adopt a conformationexhibiting some A-form features as indicated by a strong band in thevicinity of either 260 nm or 205 nm in their CD spectrum. Moreimportantly, the size of their major grooves lies intermediate betweenthe widths and depths of an A-form major groove, and a B-form majorgroove.

This example demonstrates the ability of F-neo (42a) to discriminatedifferent nucleic acids based on their propensity toward the A-formconformation. The F-neodimer (79) for example can be used todiscriminate different nucleic acids based on their propensity towardthe B-form conformation. Since DNA conformations vary from A to B forms,any DNA sequence can now be targeted by aminoglycoside (monomer/dimer)based probes disclosed herein.

Example 11 Detection of Major Groove Specific Substrates

Disclosed are fluorescent probes that bind specifically to the majorgroove of DNA. The probes bind specifically to the major groove are onlycompetitively displaced by molecules that also bind in the major groove,showing little displacement of the probe by molecules that bind in theminor groove. These probes are ideally suited to screen for other drugsand proteins that bind DNA in the major groove.

A limited number of natural products have been known to interact in themaj or groove; however the major contributor to the DNA binding of thesecompounds has been the intercalating moiety or alkylation of thenucleophilic sites on DNA. More recent emergence of carbohydratescaffolds such as neomycin and neomycin-neomycin dimer as reversible major groove binding molecules has presented new pharmacophores for DNAbinding drugs (Hamilton & Arya, 2012). Fluorescent probes have beendesigned and synthesized using the aminoglycoside scaffolds that bindspecifically in the major groove. These probes can be used as tools toscreen for other molecules that interact specifically with the majorgroove of DNA or RNA. In order to demonstrate the ability of theseprobes to discriminate from major and minor groove binding molecules wepresent the results of a displacement assay using the fluorescent probeof thiazole orange conjugated to neomycin (TO-neo (36a)) bound to theDNA sequence d(G₄C₄)₂ with a minor groove binding compound 59 (Dervan,2001).

FIG. 14 shows that the fluorescence of TO-neo (36a) increases uponbinding to DNA. The fluorescence of the probe decrease approximately 60%with the addition of the neomycin-anthraquinone 67, a synthetic moleculepredicted to bind in the major groove. However, with the addition of theminor groove binding compound 59, the change in fluorescence is lessthan 10%. The change in the fluorescence observed upon the addition ofthe compound 59 is approximately the same as that observed in the DMSOcontrol.

Additionally, FIG. 15 shows results of a displacement assay using thefluorescent probe of fluorescein conjugated to neomycin (F-neo (42a))bound to the DNA sequence d(G₄C₄)₂ with the minor groove bindingcompound 59, demonstrate that the F-neo (42a) probe is not displaced bya molecule that binds in the minor groove. Upon binding to the DNA thefluorescence of F-neo (42a) is quenched. With the addition of one molarequivalent of neomycin-anthraquinone 67, we observe a significantincrease in the fluorescence. This increase is not observed with theaddition of the minor groove binding compound 59, in which the change inthe fluorescence observed is approximately the same as that of the DMSOcontrol.

Example 12 Quadruplex DNA Assays

Disclosed are fluorescent probes that bind to quadruplex DNA. The probesthat bind are competitively displaced by molecules that bind toquadruplex DNA. These probes are ideally suited to screen for otherdrugs and proteins that bind to quadruplex DNA. We provide here thedetailed experiment the HTS by the displacement of a thiazoleorange-neomycin (TO-neo, Scheme 5) (36a) bound to a synthetic DNAd(G₄T₄G₄)₂ (SEQ ID No. 5), which forms a DNA-quadruplex, by thesynthetic molecule neomycin anthraquinone 67 (using a 96 well plateformat and a fluorescent plate reader.

Over the last two decades, the ability of guanine rich nucleic acids toform four stranded structures and inhibit the functions of telomerasehave generated immense interest in their biological functions (Zahler,Williamson, Cech, & Prescott, 1991). Since the structural elucidationsof tetramolecular, bimolecular and unimolecular quadruplexes in early90's, significant attention has been paid to develop small moleculesthat selectively recognize G-quadruplex nucleic acids with highspecificity and affinity (Monchaud et al., 2008). A number of smallmolecules, most of which have extended planar aromatic ring systems,have been reported to bind to G-quadruplexes, with the majority havingmicromolar affinities (Pagano, Mattia, & Giancola, 2009). Most of theseligands primarily recognize G-quadruplexes through terminal stackinginteractions between the i-ring systems of the ligand and the planartetrad formed by a cyclic array of four guanosines. Groove recognition,however, also presents an enticing approach for selective recognition ofquadruplex structures (Stephen, 2012). Studies have shown that thebinding of aminoglycosides to DNA-quadruplexes suggest that quadruplexgrooves are the binding domains of these ligands (Ranjan, Andreasen,Kumar, Hyde-Volpe, & Arya, 2010).

Neomycin binds to a dimeric form of a parallel quadruplex formed by theTetrahymena telomere with nanomolar affinity. This binding is thehighest affinity reported for a completely non-planar molecule bindingto a quadruplex. Disclosed are probes using fluorescent moleculesconjugated to neomycin as a tool for screening other high affinity andpotential drug candidates that bind to quadruplex DNA.

The high throughput screening (HTS) has been designed to determine thebinding of drugs to the quadrulplex DNA d(G₄T₄G₄)₂. This HTS involvesthe displacement of TO-neo (36a) by quadruplex binding molecules in a 96well plate format using a fluorescent plate reader. Initial studies ofthis system have demonstrated that the binding of thiazole orange (TO)results in flourescence of the fluorophore, and the displacement of TOby a competitive binding molecule results in the loss of fluorescence.The current work shows the same is true of the conjugated TO-neo (36a)molecule. The quality of the assay was determined by the calculation ofa Z′-factor using equation 1 for the displacement TO-neo (36a) from thequadruplex DNA by neomycin-anthraquinone 67. The final assay resultsshow in FIG. 16 were obtained using the average (μ_(n)) and standarddeviation (σ_(n)) from 24 wells of 1.0 μM F-neo (42a)/DNA d(G₄T₄G₄)₂ asthe negative control and from the average (μ_(p)) and standard deviation(σ_(p)) 24 wells of 1.0 μM F-neo (42a)/DNA d(G₄T₄G₄)₂ mixed with 1.0 μMneomycin-anthraquinone 67 as the positive control. The experimentresulted in Z′-factor of 0.78. These results indicate that the assay issuitable for the detection of drugs that bind to the DNA quadruplexes bythe displacement of the TO-neo (36a) probe in a high through-put format.The TO-neo (36a) probes can be extended to other aminoglycosidescaffolding, other fluorescent molecules, as well as other DNA that formquadruplexes.

Example 13 Synthesis and Characterizations Synthesis ofNeomycin-Thiazole Orange Conjugates

The Thiazole orange was synthesized using a procedure reported in theliterature (Yang, P. et al. (2009). In our case, we changed the linkerlength to a short alkyl chain (5 carbon) than a longer chain (10 carbon)reported before (scheme 10). Thus a carboxyl group present in thisthiazole orange derivative can be used towards coupling reactions.

An example of coupling of aminosugar (neomycin) with thiazole orangecarboxylic acid is given in scheme 11. Hexa-N-Bocdeoxy-neomycin-5″-amine 3 or 6 was reacted with thiazole orange-COOHconjugate in the presence of a coupling reagent (EDC). This leads to theformation of the Boc protected neomycin-thiazole orange conjugate whichcan be isolated after purification with column chromatography. The Bocprotecting groups in the conjugate deprotected using trifluoroaceticacid to afford neomycin-thiazole orange conjugate as their TFA salt.

Synthesis of Neomycin-TO Conjugate (36a, Linker 1)

Synthesis of 3-methyl-2-(methylthio)benzo[d]thiazol-3-ium (32)

To a dry 100 mL round bottom flask was added3-methylbenzothiazole-2-thione (2.0 g, 11.0 mmol) and iodomethane (3.2g, 23.0 mmol, 2.0 equival). The reaction mixture was heated at 50° C.for 4 h under the atmosphere of argon. The reaction mixture turned intowhite precipitate. The reaction mixture was cooled back to roomtemperature. The obtained solid was suspended in MeOH (100.0 mL).Diethyl ether was (75.0 mL) was added to induce precipitation. Theprecipitate was collected via vacuum filtration and the solid obtainedwas washed with diethyl ether (3×10.0 mL). It was then dried undervacuum to give the desired product as white solid (3.0 g, 84%): ¹H NMR(500 MHz, DMSO) δ 3.13 (s, 3H, SCH₃), 4.11 (s, 3H, NCH₃), 7.70-7.77 (m,1H, Ar), 7.82-7.88 (m, 1H, Ar), 8.20 (d, J=8.42 Hz, 1H, Ar), 8.41 (d,J=8.06 Hz, 1H, Ar).

Synthesis of 1-(4-carboxybutyl)-4-methylquinolinium (34)

To a dry round bottom flask was added 4-methylquinoline (2.0 g, 14 mmol)and 5-bromovaleric acid (2.8 g, 15.0 mmol, 1.1 equival). and Thecontents were heated at 110° C. for 3 h under argon atmosphere. Theresulting residue was dissolved in MeOH (20.0 mL) and the product wasprecipitated by addition of diethyl ether (40.0 mL). The precipitate wascollected via vacuum filtration and washed with diethyl ether (3×10.0mL). The obtained solid was dried under vacuum to give the desiredcompound as pink solid (2.2 g, 38%): ¹H NMR (500 MHz, DMSO-d₆) δ 1.62(p, J=7.0 Hz, 2H, CH₂), 1.90 (p, J=7.3 Hz, 2H, CH₂), 2.29 (t, J=6.9 Hz,2H, CH₂), 3.00 (s, 3H, CH₃), 4.63 (t, J=6.9 Hz, 2H, NCH₂), 8.04-8.10 (m,2H, Ar), 8.22-8.30 (m, 1H, Ar), 8.51-8.63 (m, 2H, Ar), 9.41 (d, J=6.23Hz, 1H, Ar).

Synthesis of(Z)-1-(4-carboxybutyl)-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolinium(35)

To a dry round bottom flask were added compound 34 (1.0 g, 2.4 mmol) andcompound 32 (0.8 g, 2.4 mmol) then dry EtOH (24.0 mL). The resultingmixture was added with dry Et₃N (0.7 mL, 5.2 mmol, 2.2 eq) whichresulted in an immediate deep red coloration of the reaction mixture.The reaction mixture was heated at 55° C. for 2 h and then at roomtemperature for 1 h under an argon atmosphere. The mixture was allowedto cool back to room temperature. Precipitation was induced by additionof diethyl ether (50.0 mL). The crude solid was suspended in acetone(70.0 mL)/diethyl ether (100.0 mL) for 1 h and then the precipitatedsolid was collected via vacuum filtration. The product was then washedwith diethyl ether (3×20.0 mL). The mixture was dried under reducedpressure to give the desired product as a red solid (250 mg, 24%): ¹HNMR (300 MHz, DMSO-d₆) δ 1.62 (p, J=7.0 Hz, 2H, CH₂), 1.90 (p, J=7.3 Hz,2H, CH₂), 2.29 (t, J=6.9 Hz, 2H, CH₂), 4.03 (s, 3H, CH₃), 4.64 (t, J=6.9Hz, 2H, NCH₂), 6.97 (s, 1H, CH), 7.38-7.49 (m, 2H, Ar), 7.62 (t, J=6.9Hz, 1H, Ar), 7.70-7.90 (m, 2H, Ar), 7.97-8.03 (m, 1H, Ar), 8.06 (d,J=7.5 Hz, 1H, Ar), 8.17 (d, J=6.9 Hz, 1H, Ar), 8.67 (d, J=7.2 Hz, 1H,Ar), 8.81 (d, J=8.4 Hz, 1H, Ar), 8.85-9.15 (br, 1H, COOH); UV (H₂O)λ_(max)=503 nm. 8503=8833.8 M⁻¹ cm⁻¹.

Synthesis of 36a

To a solution of Hexa-N-Boc deoxy-neomycin-5″-amine (30.0 mg, 24.0 μmol)in dry DMF (3.0 mL), TBTU (30.8 mg, 96.0 μmol, 4.0 equival.) was added,followed by the addition of DIPEA (24.8 mg, 0.2 mmol, 8.0 equival.) and(Z)-1-(4-carboxybutyl)-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolinium35 (14.0 mg, 32.0 μmol). The reaction mixture was stirred at roomtemperature under argon. The progress of the reaction was monitored byTLC. A new deep red colored spot was appeared on TLC which was ninhydinactive. The volatiles were dried under reduced pressure. The crudemixture was purified using column chromatography on a silica gel columnusing dichloromethane-methanol as eluent [0 to 15% EtOH in DCM (v/v)],The desired product was obtained as deep red solid (25.4 mg, 65%):R_(f)=0.42[10% MeOH in dichloromethane (v/v)]; ¹H NMR (500 MHz,CD₃COCD₃) δ 8.78-8.90 (m, 1H, Ar), 8.18 (d, J=8.51 Hz, 1H, Ar),8.00-8.12 (m, 2H), 7.85-7.98 (m, 3H, Ar), 7.76-7.84 (m, 4H, Ar), 7.68(s, 1H, Ar), 7.01 (m, 2H), 6.54 (d, J=6.57 Hz, 1H, NH), 6.40 (br, s, 1H,NH), 6.25 (d, J=8.12 Hz, 1H, NH), 5.98-6.20 (m, 2H, NH), 5.04-5.20 (m,1H), 4.90-5.02 (m, 2H), 4.60-4.90 (m, 4H), 4.40-4.60 (m, 6H), 3.98-4.30(m, 12H), 3.70-3.92 (m, 4H), 3.55-3.70 (m, 4H), 3.30-3.55 (m, 4H),3.20-3.34 (m, 2H), 1.80-1.86 (m, 1H), 1.60-1.78 (m, 1H), 1.30-1.60 (m,54H, 6×Boc protons); MS (MALDI-TOF) m/z calcd. for C₇₆H₁₁₆N₉O₂₅ (M⁺)1586.84. found 1586.30 [M]₊; UV (DCM) λ_(max)=508 nm.

To this solution of N-Boc neomycin thiazole orange conjugate (8.0 mg,5.0 μmol) in DCM (1.0 mL), TFA (0.1 mL) was added and the reactionmixture was stirred at room temperature for 3 h in dark. The progress ofthe reaction was monitored by TLC. Water (2 mL) was added to thereaction mixture and then it was washed with dichloromethane (3×3 mL).The aqueous layer was lyophilized to give the desired product (36a) as adark red solid (4.6 mg, 75%): MS (MALDI-TOF) m/z calcd. forC₄₆H₆₈N₉O₁₃S₂ (M+H₂O⁺), 1004.47. found 1004.28 [M]⁺; UV (H₂O)λ_(max)=508 nm. ε₅₀₃=9960 M⁻¹ cm⁻¹.

Synthesis of Neomycin-TO Conjugate (36b, Linker 2)

To a solution Hexa-N-Boc deoxy-neomycin-5″-amine (15.3 mg, 12.0 mol) indry DMF (3.0 mL), TBTU (15.4 mg, 48.0 μmol, 4.0 mol equival.) was added,followed by the addition of DIPEA (12.4 mg, 96.0 μmol, 8.0 mol equiv)and 35 (7.0 mg, 24.0 μmol). The reaction mixture was stirred at roomtemperature under argon which resulted in new deep red colored spot onTLC plate that was also ninhydin active. The volatiles were evaporatedunder reduced pressure. The crude product was purified using columnchromatography on silica gel using dichloromethane-ethanol (0 to 15%EtOH in DCM) as eluent. The desired product was obtained as deep redsolid (17 mg, 82%): R_(f)=0.38[10% MeOH in DCM (v/v)]; ¹H NMR (500 MHz,CD₃COCD₃) δ 8.85-8.94 (d, J=7.09 Hz, 1H, Ar), 8.70-8.77 (d, J=8.51 Hz,1H, Ar), 8.56-8.66 (br, s, 1H), 8.24 (d, J=8.52 Hz, 1H, Ar), 8.11 (d,J=7.57 Hz, 1H, Ar), 8.05 (t, J=7.41 Hz, 1H, Ar), 7.71-7.81 (m, 2H, Ar),7.61-7.68 (m, 2H, Ar), 7.55 (m, J=6.46 Hz, 1H, Ar), 7.01 (m, 2H),6.45-6.55 (br, s, 1H, NH), 6.37 (d, J=8.83 Hz, 1H, NH), 6.30 (d, J=8.20Hz, 1H, NH), 6.22 (d, J=9.62 Hz, 1H, NH), 5.86-5.94 (br, s, 1H), 5.11(d, J=5.68 Hz, 1H), 5.06 (br, s, 1H), 5.03 (br, s, 1H), 4.74-4.84 (m,2H), 4.26-4.32 (m, 1H), 4.18-4.23 (m, 1H), 4.09-4.18 (m, 4H), 4.01-4.08(m, 2H), 3.97 (t, J=9.46 Hz, 1H), 3.76-3.91 (m, 4H), 3.47-3.72 (m, 12H),3.27-3.45 (m, 4H), 3.16-3.44 (m, 2H), 3.05-3.14 (m, 4H), 2.57-2.66 (m,2H), 2.31-2.50 (m, 3H), 1.93 (p, J=2.21 Hz, 1H), 1.83 (p, J=6.94 Hz,1H), 1.36-1.61 (m, 54H, 6×Boc protons); MS (MALDI-TOF) m/z calcd. forC₇₈H₁₂₀N₉O₂₅S₂ 1646.78 found 1646.67 [M]⁺. . . UV (DCM) λ_(max)=508 nm.

To a solution of N-Boc neomycin-thiazole orange (8.2 mg, 5.0 μmol) inDCM (1.0 mL), TFA (0.1 mL) was added and the reaction mixture wasstirred at room temperature for 3 h in dark. The progress of thereaction was monitored by TLC. Water (2.0 mL) was added to the reactionmixture. It was then washed with dichloromethane (3×3.0 mL). The aqueouslayer was lyophilized to give the desired compound 36b as dark red solid(4.9 mg, 80%): ¹H NMR (500 MHz, D₂O) δ 9.22 (d, J=5.99 Hz, 1H), 8.44 (d,J=8.99 Hz, 1H), 8.37 (d, J=8.51 Hz, 1H), 8.21 (d, J=8.04 Hz, 1H), 8.11(d, J=8.67 Hz, 1H), 8.03 (d, J=8.20 Hz, 1H), 7.96 (t, J=8.04 Hz, 1H),7.81-7.88 (m, 3H), 7.06-7.16 (m, 1H), 6.99 (t, J=6.78 Hz, 1H), 6.82 (d,J=5.84 Hz, 1H), 6.60-6.69 (m, 1H), 5.92-6.03 (m, 1H), 5.29-5.35 (m, 1H),5.14-5.23 (m, 1H), 5.01 (t, J=7.14 Hz, 2H), 4.93 (t, J=7.26 Hz, 1H),4.26-4.41 (m, 2H), 4.18-4.26 (m, 2H), 4.01-4.15 (m, 2H), 3.90-3.99 (m,1H), 3.78-3.90 (m, 2H), 3.67-3.76 (m, 2H), 3.57-3.67 (m, 2H), 3.33-3.42(m, 4H), 3.20-3.30 (m, 3H), 3.10-3.19 (m, 2H), 2.99-3.09 (m, 2H),2.91-2.98 (m, 2H), 2.32-2.38 (m, 1H), 2.57-2.67 (m, 1H), 2.33-2.43 (m,1H, H_(I2eq)), 2.15-2.29 (m, 2H), 1.96-2.01 (m, 1H), 8.04 (d, J=12.77Hz, 1H, H_(I2ax)), 1.58-1.74 (m, 1H); MS (MALDI-TOF) m/z calcd. forC₄₈H₇₂N₉O₁₃S₂ 1064.47 found 1064.85[M+H₂O]⁺; UV (H₂O) λ_(max)=508 nm.ε₅₀₃=13835 M⁻¹ cm⁻¹.

Synthesis of Neomycin Fluorescein Conjugates

Fluorescein-neomycin conjugates 42a-b were prepared by coupling anactivated fluorescein ester 41a with neomycin amine 3 (Scheme 12a) orcoupling of neomycin amine 3 with fluorescein isothiocyanate 41b (scheme12b). The resulting Boc protected neomycin-fluorescein conjugate waspurified using column chromatography on silica gel which was thenfollowed by deprotection of Boc groups using trifluoroacetic acid toafford the desired compounds 42a-b in good in good yields.

Synthesis of Neomycin-Fluorescein Conjugate (42a, Linker 1)

To a DMF solution (1 mL) of fluorescein succinimidyl ester 41a (5 mg,0.0086 mmol) and 4-dimethyl-aminopyridine (catalytic amount) was addedBoc protected neomycin amine 3 (10 mg, 0.0078 mmol) and stirredovernight at room temperature. The reaction mixture was concentratedunder vacuum. Flash chromatography of the residue (8% (v/v) CH₃OH inCH₂Cl₂) yielded the Boc protected conjugate as a yellow solid (10 mg):R_(f) 0.21 (10% (v/v) CH₃OH in CH₂Cl₂). The product was then dissolvedin CH₂Cl₂ (3 mL) and trifluoroacetic acid (0.5 mL) was added to it. Themixture was stirred at room temperature for 3 h. After removing thesolvents under vacuum, the residue was dissolved in H₂O (2 mL) andextracted with dichloromethane (3×2 mL). The aqueous layer wasconcentrated under vacuum to yield the desired product (7 mg, 45% fortwo steps): UV-Vis (H₂O); λ=232, 455, 478 nm; c (478 nm)=13,260 M⁻¹cm⁻¹; ¹H NMR (500 MHz, D₂O) J=9.3 Hz, 2H), 7.33-7.32 (d, J=8.20 Hz, 1H),7.23 (s, br, 1H), 7.07-7.05 (d, J=9.36 Hz, 1H), 6.74-6.73 (d, J=6.73 Hz,1H), 5.96-5.94 (m, 1H) (ring I-1′), 5.32-5.29 (br, 2H) (contains ringIII-1″), 5.19 (s, br, 1H)(ring IV-1′″), 4.33-4.17 (m, 4H)(contains ringIII-2′″, ring IV-3″), 4.01-3.97 (t, J=9.67 Hz, 1H), 3.93-3.89 (t, J=10.3Hz, 1H), 3.83-3.78 (m, 2H), 3.71 (s, br, 1H), 3.61-3.57 (t, J=9.89 Hz,1H), 3.49-3.11 (m, 16H)(contains ring IV-2′″, ring II 1′, ring II-3′),3.07-2.99 (m, 3H), 2.87-2.84 (m, 2H), 2.73-2.61 (m, 2H), 2.55-2.53 (t,J=6.52 Hz, 2H), 2.39-2.37 (br, 1H)(ring II, 2 eq), 1.83-1.76 (m,1H)(ring II-2_(ax)). MS (MALDI-TOF) calcd m/z for C₅₀H₆₈N₈O₁₉S₂,1167.26. found 1171.86 [M+Na]⁺.

Synthesis of Neomycin-Fluorescein Conjugate (42b, Linker 2)

To a solution of Boc-protected neomycin amine 3 (15 mg, 0.012 mmol) indry pyridine (5 mL), fluorescein isothiocyanate 41b (5.4 mg, 0.013 mmol)was added with a catalytic amount of 4-dimethylaminopyridine (DMAP). Theflask was purged with argon and covered in aluminum foil. The reactionwas allowed to stir for 18 h and was monitored via TLC, which indicatedformation of the product. Pyridine was dried under reduced pressure. Thecrude reaction mixture was purified by column chromatography usinggradients of dichloromethane-methanol as eluent to afford the desiredconjugate as an orange solid. R_(f)=0.29 (90:10 DCM:MeOH v/v); IR (cm⁻¹)3358 (N−H), 2973, 2927 (C−H), 1691 (C═O), 1518 (aromatic C-H); ¹H NMR(500 MHz, CD₃COCD₃): δ 8.71 (s, 1H), 8.19 (s, 2H), 8.03 (d, J=2.09 Hz,1H), 7.19 (d, J=1.88 Hz, 2H), 6.82-6.64 (m, 8H), 6.33-6.04 (m, 4H),5.17-5.12 (m, 1H), 5.04 (s, 1H), 4.37 (s, 1H), 4.34-4.26 (m, 3H),3.74-3.32 (m, 9H), 2.84 (s, 16H), 1.48-1.31 (m, 54H) MS (MALDI-TOF) m/zcalcd for C₇₆H₁₁₀N₈O₂₉S₂ 1663.85. found 1685.76 [M+Na]⁺. TheBoc-protected conjugate (13 mg, 0.008 mmol) was dissolved indichloromethane (2.5 mL) followed by addition of trifluoroacetic acid(0.4 mL). The mixture was covered with aluminum foil and stirred for 12hours at room temperature. Water (2.0 mL) was added to the solutionfollowed by washes with dichloromethane (3×1 mL). The aqueous layer waslyophilized to give the desired product as yellowish solid (quant).

Synthesis of Neomycin-Methidium Conjugate (52)

Neomycin methidium conjugate was prepared via formation of an amidebond. Commercially available methidium carboxylic acid 51 was coupledwith Boc protected neomycin amine 3 using DCC/DMAP as coupling agentwhich led to the formation of the Boc protected neomycin-methidiumconjugate that can be purified using column chromatography on a silicagel. The Boc protecting groups were then deprotected usingtrifluoroacetic acid to afford the desired conjugate 52 as theirtrifluoroacetate salt (scheme 13).

Synthesis Procedure and Characterization of Neomycin-Methidium Conjugate(52)

To a solution of6-(4-carboxyphenyl)-3,8-diamino-5-methylphenanthridinium chloride (8.6mg, 0.023 mmol) in dry DMF (3.0 mL), dicyclohexylcarbodiimide (4.8 mg,0.23 mmol) and dimethylaminopyridine (1 mg, 0.01 mmol) were added. Thesolution was allowed to stir under positive N₂ gas for 3 h. A solutionof Boc-neomycin amine 3 (30 mg, 0.023 mmol) in dry DMF (3.0 mL) wasadded via cannula. The reaction was allowed to stir at room temperatureunder positive N₂ for 28 h. The volatiles were removed in vacuo. The drysolid was washed with CH₂Cl₂. The resulting solid was dried in vacuo.Flash chromatography (0%-25% MeOH:CH₂:Cl₂) afforded Boc protected 52(30.8 mg, 80%) as a purple solid: R_(f) 0.2 in 85:15 CH₂Cl₂:MeOH); UVmax (95% CH₃OH) 286, 510 nm; IR (CHCl₃) 3405 (C-OH), 1600 (amide C═O)cm⁻¹; ¹H NMR (500 MHz, methanol-d₄, 25° C.) δ) 5 (500 MHz, J=9.1 Hz),8.56 (d, 1H, J=9.1 Hz, H10), 7.90 (d, 2H, J=8.7, H18, H₂O), 7.65 (d, 2H,J=8.8, H17, H21), 7.56 (dd, 1H, J=9.1, H4), 7.35-7.39 (m, 3H, H2, H7,H9), 5.41 (br, 1H), 4.92 (s, 1H), 4.34 (br, 2H), 4.04 (s, 1H), 4.00-3.98(m, 1H), 3.89-3.80 (m, 1H), 3.76-3.71 (m, 1H), 3.61-3.69 (m, 2H),3.55-3.52 (m, 1H), 3.51 (d, 2H), 3.47 (br, 1H), 3.4-3.2 (m, 1H), 3.19(m, 1H), 2.93-2.91 (m, 4H), 2.85-2.83 (m, 4H), 2.81 (d, 2H), 2.79-2.70(m, 2H), 1.96-1.86 (m, 1H), 1.71 (br, 1H), 1.36-1.48 (m, 54H); ¹³C NMR(125 MHz, methanol-d₄, 25° C.) δ) 5 (125 MHz, methanol-d3H, H2, H7, H9),5.41 (br, 1H), 4.92 (s, 1H), 4.34 (br, 2H), 4.04 (s, 1H), 4.00-3.98 (m,1H), 3.89-3.80 (m, 1H), 3.76-3.71 (m, 1H), 3.61-3.69 (m, 2H), 3.55-3.52(m, 1H), 3.51 (d, 2H), 3.47 (br, 1H), 3.4-3.2 (m, 1H), 3.1, 43.3, 43.2,41.7, 41.6, 33.3, 27.3; MALDI-TOF m/z (rel. intensity) calculated forC₅₁H₇₆N₁₀O₁₅SCl [M+H]⁺1600.09 found 1600.01.

To a solution of Boc protected 52 (30.8 mg, 0.018 mmol) in 3 mLdichloromethane was added trifluoroacetic acid (3 mL). 1,2-ethanedithiol(100 t-ethanedithiol (1000 in 3 mL dichloromethane was addedtrifluoroacetic acid (3 mL). 14.04 (s, 1H), 4.00-3.98 (m, 1H), 3.89-3.80(m, 1H), 3.76-3.71 (m, 1H), 3.61-3.69 (m, 2H), 3.55-3.52 (m, 1H), 3.51(d, 2H), 3.47 (br, 1H), 3.4-3.2 (m, 1H), 3.1, 43.3, 43. ratory HPLCusing a reverse phase column, (0%-100% H₂O:MeCN 0.1% TFA, 15 min). Thecompound eluted at 20.93 min. Fractions containing the compound werelyophilized affording 52 (28.0 mg, 90%) as a maroon solid: UV max (95%H₂O) 288, 514 nm; IR (KBr) 3410 (C−OH), 1605 (amide C═O) cm⁻¹; ¹H NMR(500 MHz, D₂O, 25° C.) δ) 25 (500 MHz, J=9.6 Hz, H1), 8.69 (d, 1H, J=9.6Hz, H10), 7.86 (d, 2H, J=8.7, H18, H₂O), 7.66 (d, 2H, J=8.8, H17, H21),7.52 (dd, 1H, J=9.6, H4), 7.35-7.39 (m, 3H, H2, H7, H9), 5.37 (br, 1H),5.09 (s, 1H), 4.53 (br, 2H), 4.23 (s, 1H), 4.01-3.88 (m, 1H), 3.79-3.71(m, 1H), 3.75-3.68 (m, 1H), 3.61-3.59 (m, 2H), 3.59-3.53 (m, 1H), 3.52(d, 2H), 3.47 (br, 1H), 3.29-3.21 (m, 1H), 3.17-3.03 (m, 1H), 2.91-2.89(m, 4H), 2.89-2.85 (m, 4H), 2.85 (d, 2H), 2.79 (m, 2H), 1.93 (m, 1H),1.89 (br, 1H); ¹³C NMR (125 MHz, D₂O, 25° C.) δ) 25 (125 MHz, D7.39 (m,3H, H2, H7, H9), 5.37 (br, 1H), 5.09 (s, 1H), 4.53 (br, 2H), 4.23 (s,1H), 4.01-3.88 (m, 1H), 3.79-3.71 (m, 1H), 3.75-3.68 (m, 1H), 3.61-3.59(m, 2H), 3.59-3.53 (m, 1H), 3.52 (d, 2H), 3.47 (br, 1H), 3.29-3.21 (m,1H), 3.17-3.03 (m8.7, 42.3, 40.5, 40.4, 29.3; MALDI m/z (rel intensity)calculated for C₄₆H₆₇N₁₀O₁₃SCl [M+H]⁺ 1000.07 found [M+Na]⁺1000.14.

Synthesis of Neomycin-Pyrene Conjugate (60)

Neomycin-pyrene conjugate was synthesized via the formation of an amidebond. Boc protected neomycin amine 3 was reacted with commerciallyavailable pyrene succinimidyl ester 59. Displacement of the succinimidylester group leads to the formation of the amide bond (scheme 14) andthus a Boc protected neomycin-pyrene conjugate can be prepared afterpurification using column chromatography. The Boc protected compound canbe deprotected using trifluroacetic acid to give the desired conjugate60 as a trifluoroacetate salt.

Synthesis of Neomycin Pyrene Conjugate (60)

To a solution of Boc-protected neomycin amine (15 mg, 0.013 mmol) in drypyridine (10 mL), a solution of pyrene succinimide ester (20 mg, 0.051mmol) was added with a catalytic amount of 4-dimethylaminopyridine(DMAP). The flask was purged with argon and covered in aluminum foil.The reaction was allowed to stir for 18 h at room temperature and theprogress of the reaction was monitored via TLC, which indicatedformation of the product. Pyridine was dried under reduced pressure. Thecrude reaction mixture was purified by column chromatography usinggradients of dichloromethane-methanol as eluent to afford Boc protected60 as a white solid (13 mg, 66%): ¹H NMR (300 MHz, CD₃COCD₃) δ 8.47 (d,J=9.82 Hz, 1H), 8.33-8.24 (m, 3H), 8.22 (d, J=2.13 Hz, 1H), 8.17-7.97(m, 4H), 6.56-6.05 (m, 3H), 5.15 (m, 1H), 5.04 (s, 1H), 4.26 (d, J=4.38Hz, 2H), 4.16 (s, 1H), 3.98-3.74 (m, 3H), 3.70-3.34 (m, 9H), 3.30-2.58(m, 9H), 2.43 (t, J=7.18 Hz, 1H), 2.29-2.20 (m, 2H), 1.65-0.95 (m, 41H).

The Boc-protected 60 (13 mg, 0.008 mmol) was dissolved indichloromethane (2.5 mL) followed by addition of trifluoroacetic acid(0.4 mL). The mixture was covered with aluminum foil and stirred for 12h at room temperature. Water (2.0 mL) was added to the solution followedby washes with dichloromethane (3×1 mL). The aqueous layer waslyophilized to give the desired product 60 as yellowish solid (6.75 mg,82%): NMR (300 MHz, CD₃COCD₃) δ 8.13-8.03 (m, 3H), 8.01-7.86 (m, 5H),7.34 (d, J=8.16 Hz, 1H), 5.82 (d, J=4.08 Hz, 1H), 5.21 (d, J=3.71 Hz,1H), 4.16-3.67 (m, 10H), 2.63 (s, 2H), 3.23-2.74 (m, 12H), 2.53-2.21 (m,7H), 2.08 (d, J=5.56 Hz, 1H), 2.06-1.95 (m, 3H), 1.76 (d, J=12.98 Hz,1H); MS (MALDI-TOF) m/z cacld. for C₄₅H₆₅N₇O₁₃S 944.10. found 966.90[M+Na]⁺; UV (water): λ_(max1) (nm)=239, λ_(max2) (nm)=274, λ_(max2)(nm)=342.

Synthesis of Neomycin Dimer Thiazole Orange Conjugate (63)

For the synthesis of neomycin dimer-thiazole orange conjugate 63, firsta linker 61 was constructed that had a pendant amine group for couplingto thiazole orange carboxylic acid which leads to the formation of 62.The Boc protecting groups in 62 can then be deprotected usingtrifluoroacetic acid and subsequently coupled with Boc protectedneomycin isothiocyanate 4, to form Boc protected neomycin dimer-thiazoleorange conjugate 63. The Boc groups can then be deprotected to give thedesired neomycin dimer-thiazole orange conjugate as trifluoroacetatesalt (scheme 15).

Synthesis and Characterization of Thiazole Orange-Neomycin Dimer (63)

To a solution of neomycin dimer amine (53) (14.6 mg, 6.0 μmol) in dryDMF (2.0 mL), in dry DMF (3.0 mL), TBTU (7.9 mg, 24.0 μmol, 4.0 molequival.) was added, followed by the addition of DIPEA (6.2 mg, 48.0μmol, 8.0 mol equival.) and thiazole orange-COOH (35) (3.5 mg, 12.0μmol). The reaction started in the atmosphere of argon at r.t. withconstant stirring. The progress of the reaction was monitored by TLC.The reaction ran overnight in inert atmosphere in dark. The volatileswere removed on roto-vap. Column chromatography (0 to 25% EtOH in DCM)results in an orange solid (8.6 mg). [R_(f) 0.24, 13% MeOH in DCM(v/v)].

The reaction mixture was then dissolved in DCM (1.0 mL) followed byaddition of TFA (0.1 mL). The mixture was stirred at room temperatureunder darkness for 3 h during which the progress of the reaction wasmonitored by TLC. Water (2.0 m T) was added to the reaction mixture andthen washed with DCM (3×3.0 mL). The water layer was lypholyzed thatresults in a dark red coloured compound 63 (6.05 mg, 20% overall yieldfor the two steps). ¹H NMR (500 MHz, D₂O) δ 9.23 (m, 1H), 8.44 (d,J=8.84 Hz, 1H), 8.39 (d, J=8.36 Hz, 1H), 8.21-8.24 (m, 1H), 8.11-8.14(m, 1H), 8.02 (d, J=8.20 Hz, 1H), 7.98 (t, J=8.04 Hz, 1H), 7.82-7.89 (m,3H), 7.63-7.72 (m, 2H), 7.40-7.52 (m, 4H), 5.91-6.04 (m, 2H), 5.29-5.35(m, 2H), 5.15-5.22 (m, 2H), 5.02 (t, J=7.24 Hz, 2H), 4.29-4.40 (m, 4H),4.18-4.26 (m, 4H), 4.05-4.17 (m, 4H), 3.80-3.96 (m, 6H), 3.55-3.73 (m,4H), 3.20-3.55 (m, 20H), 2.95-3.20 (m, 4H), 2.55-2.78 (m, 2H), 2.35-2.45(m, 2H, H_(I2eq)), 2.18-2.30 (m, 2H), 1.97-2.13 (m, 2H), 1.87 (d,J=12.84 Hz, 2H, H_(I2ax)), 1.60-1.75 (m, 2H); MS MALDI-TOF calcd. forC₈₇H₁₄₉N₂₀O₂₅S₅ (M⁺), 2035.56, obsd: 2036.122. UV (H₂O) λ_(max)=508 nm.ε₅₀₃=13835 M⁻¹ cm⁻¹.

Synthesis of Neomycin-Naphthalenediimide Conjugate (65)

A monoamine terminated naphthalene diimide can be prepared in fewsynthetic steps as described earlier. The amine terminatednaphthalenedimide 64 can be reacted with Boc protected neomycinisothiocyanate 4 to form a thiourea linked Boc neomycin-naphthalenediimide conjugate (scheme 16) after purification using columnchromatography. The Boc groups can then be deprotected usingtrifluoroacetic acid to give the desired conjugate 65 astrifluoroacetate salt.

Synthesis of Neomycin-Napthalenediimide Conjugate (65)

Preparation of Boc Protected Neomycin-Napthalenediimide Conjugate

To an anhydrous pyridine solution (2 mL) of Boc protected neomycinisothiocynate 4 (18 mg, 0.014 mmol) were added amine terminatednapthalenediimide 64 (7.6 mg, 0.015 mmol) and DMAP (catalytic amount).After the mixture had been stirred under N₂ at room temperatureovernight, the organic solvent was removed under vacuum. Flashchromatography of the residue (6% MeOH in CH₂Cl₂) yielded the desiredproduct 65 as a white solid (15 mg, 86%): R_(f)=0.33 (silica gel, 6%MeOH in CH₂Cl₂); ¹H NMR (CD₃OD) δ 8.74-8.77 (m, 4H), 5.40 (br, 1H), 5.37(m, 1H), 5.11 (m, 1H), 4.90 (m, 1H), 4.23 (m, 1H), 4.09 (m, 2H), 3.82(m, 4H), 3.76 (m, 2H), 3.67 (m, 2H), 3.44-3.56 (m, 6H), 3.00-3.30 (m,9H), 2.84-2.86 (m, 4H), 2.70-2.78 (m, 6H), 2.69 (m, 4H), 2.35 (m, 6H),2.03 (m, 2H), 1.94 (m, 1H), 1.40 (m, 54H); MS (MALDI-TOF) m/z calcd forC₇₇H₁₁₉N₁₁O₂₈S₂Na, 1610.96. found 1611.56.

Preparation of Neomycin Napthalenediimide Conjugate (65)

In a 10 mL round-bottom flask, Boc protected neomycin napthalenediimideconjugate (15 mg, 0.090 mmol) was dissolved in a 1:1 TFA/CH₂Cl₂ mixture(2 mL) and the solution stirred at room temperature for 3 h. The solventwas removed under vacuum, and the residue was dissolved in deionizedwater (20 mL). The aqueous layer was washed with ether (3×20 mL).Aqueous layer was lyophilized which yielded 3 as a yellow solid (9 mg,90%): ¹H NMR (CD₃OD) δ 8.74-8.77 (m, 4H), 5.40 (br, 1H), 5.37 (m, 1H),5.11 (m, 1H), 4.90 (m, 1H), 4.90 (q, 2H) 4.23 (m, 2H), 4.09 (m, 2H),3.82 (m, 6H), 3.76 (m, 6H), 3.67 (m, 2H), 3.44-3.56 (m, 6H), 3.00-3.30(m, 12H), 2.84-2.86 (m, 6H), 2.70-2.78 (m, 8H), 2.69 (m, 4H), 2.45 (m,2H), 2.03 (m, 2H), 1.94 (m, 1H), 1.45 (m, 2H).

Synthesis of Neomycin-Anthraquinone Conjugate (67)

To prepare neomycin anthraquinone conjugate, anthraquinoneisothiocyanate 66 was reacted with neomycin amine 3 in the presence ofDMAP catalyst. This leads to the formation of the Boc protectedneomycin-anthraquinone conjugate which can be isolated pure afterpurification using column chromatography (scheme 17). The Boc protectedconjugate can be deprotected using trifluoroacetic acid to give thedesired conjugate 67 as trifluoroacetate salt

Preparation of Neomycin-Anthraquinone Conjugate (67)

a. Preparation of Boc Protected Neomycin-Anthraquinone Conjugate

To an anhydrous pyridine solution (5 mL) of compound 12 (10.7 mg, 0.008mmol) were added compound 6 (9.3 mg, 0.027 mmol) and DMAP (catalyticamount). After the mixture had been stirred at room temperatureovernight, the organic solvent was removed under vacuum. Flashchromatography of the residue (5% MeOH in CH₂Cl₂) yielded the desiredproduct as a white solid (7.1 mg, 86%): R_(f)=0.60 (silica gel, 10% MeOHin CH₂Cl₂); ¹H NMR (CD₃OD) δ 8.60 (s, 1H), 8.20-8.40 (br, 1H), 5.37 (m,1H), 5.11 (m, 1H), 4.90 (m, 1H), 4.23 (m, 1H), 4.09 (m, 2H), 3.82 (m,4H), 3.76 (m, 2H), 3.67 (m, 2H), 3.44-3.56 (m, 6H), 3.00-3.30 (m, 9H),2.84-2.86 (m, 4H), 2.70-2.78 (m, 6H), 2.69 (m, 4H), 2.35 (m, 6H), 2.03(m, 2H), 1.94 (m, 1H), 1.40 (m, 54H); MS (MALDI-TOF) m/z calcd forC₇₄H₁₁₃N₉O₂₇S₂Na, 1646.86. found 1647.19.

b. Deprotection of Boc Protected Neomycin-Anthraquinone Conjugate

In a 10 mL round-bottom flask, Boc protected neomycin-anthraquinoneconjugate (7.1 mg, 0.090 mmol) was dissolved in a 1:1TFA/CH₂Cl₂ mixture(2 mL) and the reaction mixture was stirred at room temperature for 3 h.The solvent was removed under vacuum, and the residue was dissolved indeionized water (20 mL). The aqueous layer was washed with ether (3×20mL). Aqueous layer was lyophilized which yielded the desired compound 67as a yellow solid (9 mg, 90%): ¹H NMR (CD₃OD) δ 8.60 (s, 1H), 8.20-8.40(m, 4H), 7.8-7.84 (m, 2H), 5.40 (br, 1H), 5.37 (m, 1H), 5.11 (m, 1H),4.90 (m, 1H), 4.50 (m, 2H), 4.23 (m, 1H), 4.09 (m, 2H), 3.82 (m, 4H),3.76 (m, 2H), 3.67 (m, 2H), 3.44-3.56 (m, 8H), 3.00-3.30 (m, 10H),2.84-2.86 (m, 4H), 2.70-2.78 (m, 6H), 2.69 (m, 4H), 2.35 (m, 6H), 2.03(m, 2H), 1.94 (m, 1H); MS (MALDI-TOF) m/z calcd for C₄₄H₆₅N₉O₁₅S₂Na,1047.2. found 1046.9.

Synthesis of Kanamycin-Pyrene Conjugate (69)

Kanamycin-pyrene can be synthesized by reaction of kanamycin amine 68with pyrene succinimidyl ester 59 (scheme 18). This leads to theformation of Boc protected kanamycin-pyrene conjugate which can bedeprotected in the presence of trifluoroacetic acid to give the desiredconjugate 69.

Preparation of Boc Protected Kanamycin-Pyrene (69)

To a solution of Boc protected kanamycin amine (30 mg, 0.03 mmol) inpyridine, DMAP (catalytic) was added followed by addition of pyrenesuccinimidyl ester (1.25 mg, 0.64 mmol). The mixture was stirred at roomtemperature for 14 h. Volatiles were removed under reduced pressure. Thecrude mixture was purified on a silica gel column using ethylacetate-methanol as eluent to afford the desired conjugate (Yield=88%).R_(f)=0.66 (ethyl acetate-methanol 86:14 v/v).

Synthesis of an Amino Functionalized Neomycin Dimer (53)

A tridentate linker 76 was designed and synthesized for the preparationof dimer conjugates. Compound 76 was prepared according to procedurereported in literature in which the primary amines of spermidine wereselectively protected using tert-butyl phenyl carbonate (Phanstiel,Price, Wang, Juusola, Kline, & Shah, 2000). In the next step, thesecondary amine was reacted with 5-bromovaleronitrile throughnucleophilic substitution reaction in the presence of a base tointroduce nitrile functionality (Phanstiel, Price, Wang, Juusola, Kline,& Shah, 2000). As shown in scheme 19, the TFA salt of 76 was thenreacted with Hexa-N-Boc deoxy-neomycin-5″-isothiocyanate in astoichiometry ratio of 2:1 to form nitrile functionalized neomycindimer. The neomycin dimer with a nitrile was then reduced and convertedinto an amine functionalized neomycin dimer 53 (scheme 20), which can becoupled to fluorescent dyes.

Synthesis of Nitrile Functionalized Boc Protected Neomycin Dimer (88)

To a solution of 5-((4-aminobutyl)(3-aminopropyl)amino)pentanenitriletrifluoroacetic acid (6.8 mg, 16.0 μmol) in dry pyridine (2.0 mL),triethylamine (7.0 mg, 70.0 μmol) was added and the reaction mixturestirred for 15 min. followed by addition of Hexa-N-Bocdeoxy-neomycin-5″-isothiocyanate (4) (45.0 mg, 34.0 μmol) and thereaction started in the atmosphere of argon. The reaction was runovernight and the progress of the reaction was monitored by TLC. Thereaction mixture was then dried and column chromatography (0 to 15% EtOHin DCM) results in an off white solid 88 (36.4 mg, 79%). [R_(f) 0.31,10% MeOH in DCM (v/v)]; IR (KBr, cm⁻¹) 3388 (broad), 2977, 2150, 1667,1519, 1366, 1251; ¹H NMR (500 MHz, CD₃COCD₃): δ 6.45-6.58 (m, 4H,NH_(6IV)), 6.35-6.41 (d, J=9.45 Hz, 2H, NH), 6.28-6.35 (d, J=5.04 Hz,2H, NH), 6.07-6.27 (m, 8H, NH_(1I), NH_(3I), NH_(2IV), and NH_(2II)),5.90-6.04 (br, s, 4H), 5.14-5.32 (m, 8H), 4.81-4.91 (m, 4H), 4.63 (p,J=6.78 Hz, 4H), 4.53-4.59 (m, 4H), 4.43 (br, s, 2H), 4.16-4.33 (m, 8H),4.06 (s, 4H), 3.90-3.96 (m, 4H), 3.73-3.90 (m, 14H), 3.53-3.73 (m, 20H),3.41-3.54 (m, 10H), 3.10-3.35 (m, 18H), 3.01-3.10 (m, 4H), 2.54 (t,J=7.10 Hz, 4H), 2.20 (t, J=2.21 Hz, 2H), 1.94 (t, J=2.21 Hz, 2H),1.75-1.83 (m, 4H), 1.66-1.75 (m, 4H), 1.56-1.61 (m, 2H), 1.27-1.55 (m,114H, H_(2Ieq), 6×boc, linker protons); MS MALDI-TOF calcd. forC₁₂₄H₂₂₀N₁₈O₄₈S₄ (M+Na⁺), 2881.43, obsd: 2881.10.

Synthesis of Amino Functionalized Boc Protected Neomycin Dimer (53)

To a solution of 88 (25.0 mg, 90.0 μmol) in dry ethanol (10.0 mL),raney's nickel (20.0 mg, 50% slurry) and NH₄OH (50.0 μL) was added. Thereaction mixture stirred at 0° C. for 5 min. following which ammonia gaswas passed through the solution for 15 min. at 0° C. The solution wasthen allowed to come to r.t. and the flask was partially evacuated. Thereaction mixture was stirring in the atm. of H2 gas overnight and theprogress of the reaction was monitored by TLC. The reaction mixture wasfiltered and the solvents were evaporated. Column chromatography [0 to20% EtOH in DCM, (v/v)] results in greenish powder (15 mg, 60%). [R_(f)0.41, 15% MeOH in DCM (v/v)]; IR (KBr, cm⁻¹) 3330-3450 (broad), 2967,2959, 2855, 1696, 1526; ¹H NMR (500 MHz, CD₃COCD₃) δ 7.14 (s, 1H), 6.68(m, 1H), 5.86-6.33 (m, NH_(6IV), NH_(6II), NH_(1I), NH_(3I), NH_(2IV),and NH_(2II)), 5.09-5.39 (m, 8H), 4.98-5.08 (m, 4H), 4.75-4.96 (br, s,2H), 4.42-4.67 (m, 4H), 4.53-4.59 (m, 4H), 4.43 (br, s, 2H), 4.13-4.39(m, 10H), 3.98-4.13 (m, 8H), 3.73-3.93 (m, 16H), 3.53-3.73 (m, 22H),3.40-3.53 (m, 10H), 3.01-3.38 (m, 18H), 2.68-2.98 (m, 6H), 2.51-2.64 (m,4H), 1.83-1.91 (m, 2H), 1.76-1.83 (m, 4H), 1.69-1.75 (m, 4H), 1.16-1.67(m, 114H, H_(2Ieq), 6×boc, linker protons); MS MALDI-TOF calcd. forC₁₂₄H₂₂₄N₁₈O₄₈S₄ (M+Na⁺), 2885.43, obsd: 2886.07.

Synthesis of Neomycin Dimer-Anthraquinone Conjugate (70)

To prepare neomycin dimer anthraquinone conjugate 70, anthraquinoneisothiocyanate 37 was reacted with neomycin dimer amine 53 in thepresence of DMAP catalyst. This leads to the formation of the Bocprotected neomycin dimer-anthraquinone conjugate which can be isolatedpure after purification using column chromatography (scheme 21). The Bocprotected conjugate can be deprotected using trifluoroacetic acid togive the desired conjugate 70 as trifluoroacetate salt.

Synthesis and Characterization of Neomycin Dimer Anthraquinone (70)

To a solution of N-Boc neomycin dimer amine 53 (22.0 mg, 9.0 mol) in drypyridine (2.0 mL), anthraquinone isothiocynate 37 (2.8 mg, 10.0 mol) wasadded which was followed by addition of triethylamine (2.0 mg, 20.0μmol). The reaction mixture was stirred at room temperature overnightunder argon. The progress of the reaction was monitored by TLC. Thesolvents were evaporated under reduced pressure. The crude product waspurified using column chromatography on silica gel usingdichloromethane-methanol as eluent (0 to 10% MeOH in DCM (v/v)). Thedesired product was obtained as faint orange colored solid (22.1 mg,77%): R_(f)=0.48 (10% EtOH in DCM (v/v)); IR (KBr, cm⁻¹) 3300-3500 (br),2920, 2103 (br, —C═S), 1711, 1609; ¹H NMR (500 MHz, CD₃COCD₃) d8.20-8.35 (m, 4H aromatic hydrogens from anthraquinone), 7.90-8.01 (m,4H, aromatic hydrogens from anthraquinone), 6.38-6.45 (t, J=6.15 Hz, 2H,NH_(6IV)), 6.18-6.30 (4H, NH_(II), NH_(6II)), 6.05-6.15 (m, 4H,NH_(2IV), NH_(3I)), 5.90-6.02 (br, s, 2H, NH_(2II)), 5.25-5.30 (m, 2H),5.20-5.24 (m, 2H), 5.10-5.18 (m, 4H), 4.96-5.08 (m, 6H), 4.77-4.85 (m,2H), 4.56-4.64 (m, 2H), 4.44-4.52 (m, 2H), 4.34-4.42 (m, 2H), 4.15-4.33(m, 10H), 4.01-4.09 (m, 2H), 3.88-3.99 (m, 4H), 3.71-3.87 (m, 8H),3.40-3.70 (m, 22H), 3.20-3.31 (m, 6H), 2.96-3.06 (m, 4H), 1.68-1.76 (m,2H), 1.25-1.58 (m, 112H, 12×Boc, linker protons); MS (MALDI-TOF) m/zcalcd. for C₁₄₀H₂₃₃N₁₉O₅₀S₅, 3142.77. found 3166.18 [M+Na]⁺; UV (DCM)λ_(max)=254 nm. The Boc protected 70 was taken up in DCM-TFA solution(2.2 mL, 1:0.1 v/v) and stirred at room temperature under darkness for 2h. Progress of reaction was checked by TLC. To this, deionized water(2.0 mL) was added and the mixture was washed with DCM (2×3 mL). Theaqueous layer was lyophillized to afford the desired compound asslightly greenish white solid (22.3 mg, 90%); ¹H NMR (500 MHz, D₂O) δ)MR (500 MHz, Droom temperature under darkness for 2 h. Progress ofreaction was checked by TLC. To this, deionized water (2.0 mL) was addedand the mixture was washed with DCM (2×3 m·82 (m, 2H, aromatic hydrogensfrom anthraquinone), 7.63-7.71 (m, 1H, aromatic hydrogens fromanthraquinone), 5.91 (br, s, 2H), 5.24 (s, 2H), 5.08 (s, 2H), 4.18-4.30(m, 8H), 4.10-4.18 (m, 4H), 4.05 (m, 2H), 3.85-4.01 (m, 2H), 3.74-3.84(m, 4H), 3.60-3.73 (m, 4H), 3.15-3.61 (m, 18H), 2.90-3.17 (m, 4H),2.60-2.89 (m, 4H), 2.31-2.41 (m, 2H, H_(2Iax.)), 1.78-1.91 (m, 2H,H_(2Ieq.)), 1.02-1.30 (m, 4H); MS (MALDI-TOF) m/z calcd. forC₈₀H₁₃₇N₁₉O₂₆S₅ 1941.38. found 1960.18 [M+H₂O]⁺; UV (H₂O): λ_(max)=342nm, ε₃₄₂=99018 M⁻¹ cm⁻¹.

Synthesis of Neomycin Dimer-Pyrene Conjugate (87)

Neomycin dimer-pyrene conjugate was synthesized via formation of anamide bond. Boc protected neomycin dimer amine 53 was reacted withpyrene succinimidyl ester 38, leading to the formation of the amide bond(scheme 22) and thus a Boc protected dimer-pyrene conjugate can beisolated after purification using column chromatography. The Bocprotected compound can be deprotected using trifluroacetic acid to givethe desired conjugate 87 as a trifluoroacetate salt.

Synthesis and Characterization of Neomycin Dimer-Pyrene Conjugate (87)

To a solution of N-Boc dimer amine 53 (22.0 mg, 9.0 μmol) in drypyridine (2.0 mL), pyrene succinimide ester 38 (3.6 mg, 10.0 μmol) wasadded followed by addition of triethylamine (2.0 mg, 20.0 μmol). Thereaction mixture was stirred at room temperature overnight under theatmosphere of argon. The progress of the reaction was monitored by TLC.The solvents were evaporated under reduced pressure. The crude productwas purified using column chromatography on silica gel usingdichloromethane-methanol as eluent [0 to 10% MeOH in DCM (v/v)]. Thedesired product was obtained as off white solid (26.1 mg, 94%).:R_(f)=0.42 (10% EtOH in DCM (v/v)]; IR (KBr, cm⁻¹) 3300-3500 (br), 2975,2918, 2105 (br, —C═S), 1705, 1619; ¹H NMR (500 MHz, CD₃COCD₃) δ CDR (500MHz, CD2975, 2918, v)]; wed by addition of triethylamine (2.0 mg, 20.0μmol). The reaction mixture was stirred at r6.40-6.51 (m, 2H, NH_(6Iv)),6.30-6.36 (2H, NH_(1I) or NH_(6II)), 6.20-6.27 (m, 2H, NH_(II) orNH_(6II)), 6.05-6.20 (m, 4H, NH_(2IV), NH_(3I)), 5.82-5.97 (br, s, 2H,NH_(2II)), 5.10-5.23 (m, 4H), 4.95-5.05 (m, 4H), 4.76-4.82 (m, 2H),4.35-4.65 (m, 34H), 4.12-4.31 (m, 10H), 3.99-4.10 (m, 2H), 3.88-3.96 (m,2H), 3.75-3.88 (m, 6H), 3.38-3.70 (m, 26H), 3.05-3.30 (m, 8H), 2.75-2.85(m, 4H), 2.38-2.48 (m, 4H), 2.14-2.24 (m, 4H), 1.65-1.75 (m, 2H),1.10-1.65 (m, 112H, 12×Boc, linker proton); MS (MALDI-TOF) m/z calcd.for C₁₄₃H₂₃₈N₁₈O₄₉S₄ (M+Na⁺), 3144.76. found 3145.213; UV (DCM):λ_(max)=345 nm. The Boc protected compound was taken up in DCM-TFAsolution (2.2 mL, 1:0.1 v/v) and stirred at room temperature underdarkness for 2 h. Progress of reaction was checked by TLC. To this,deionized water (2.0 mL) was added and the mixture was washed with DCM(2×3 mL). The aqueous layer was lyophillized to afford the desiredcompound 87 as slightly greenish white solid (25.61 mg, 95%); ¹H NMR(500 MHz, D₂O) δ 8.62-8.70 (m, 1H, aromatic hydrogens from Pyrene),8.10-8.20 (m, 4H, aromatic hydrogens from Pyrene), 7.80-8.01 (m, 4H,aromatic hydrogens from Pyrene), 7.73-7.82 (m, 2H, aromatic hydrogensfrom Pyrene), 7.63-7.71 (m, 1H, aromatic hydrogens from Pyrene),5.19-5.23 (m, 4H), 4.05-4.15 (m, 4H), 3.85-4.04 (m, 4H), 3.65-3.84 (m,8H), 3.48-3.61 (m, 4H), 3.12-3.45 (m, 22H), 2.95-3.12 (m, 6H), 2.76-2.94(m, 2H), 2.26-2.50 (m, 8H, H_(2Iax.)), 1.69-1.81 (m, 2H, H_(2Ieq.)),1.02-1.30 (m, 4H); MS (MALDI-TOF) m/z for C₈₃H₁₄₂N₁₈O₂₅S₄ [M+H₂O]⁺,calcd 1938.40. found 1938.91; UV (H₂O): λ_(max)=342 nm, ε₃₄₂=99018 M⁻¹cm⁻¹.

Synthesis of Neomycin Dimer-Naphthalenediimide Conjugate (88)

A monoamine functionalized naphthalene diimide 64 can be prepared in fewsynthetic steps as described earlier. The isothiocyanate terminatednaphthalenedimide 39 was reacted with Boc protected spermidine amine 61to form a thiourea linked naphthalene diimide conjugate 88a (scheme 23)after purification using column chromatography. The Boc groups weredeprotected using trifluoroacetic acid and the amine reacted withneomycin isothiocyanate 4 to give the desired conjugate 88 astrifluoroacetate salt.

Neomycin Dimer Naphthalenediimide (88)

Synthesis of Spermidine N-Boc Naphthalenediimide (88a)

To a solution of Boc protected spermidine amine (61) (15.0 mg, 0.04mmol) in pyridine (3.0 mL) a catalytic amount of DMAP, andNaphthalenediimide isothiocyanate (39) (34.1 mg, 0.10 mmol) were added,followed by stirring at room temperature overnight. Volatiles wereevaporated under reduced pressure and the crude product was purified bycolumn chromatography on silica gel using DCM-MeOH (0-15% MeOH in DCM)as eluent to afford desired compound 88a as off white solid (6.4 mg,22%); R_(f)=0.32[10% EtOH in DCM (v/v)]; ¹H NMR (500 MHz, CD₃COCD₃) δ8.78 (m, 4H), 4.22-4.38 (m, 2H), 3.50-3.70 (m, 3H), 3.21-3.38 (m, 2H),3.08-3.21 (m, 2H), 2.29-2.38 (m, 2H).

Synthesis of Neomycin Dimer Naphthalenediimide Conjugate (88)

The Boc protected napthalenediimide derivative 88a from the previousstep was taken up in DCM-TFA (2.0 mL, 1:1 v/v) and stirred at roomtemperature for 4 h. Volatiles were removed under reduced pressure andthe residue was taken up in pyridine (3.0 mL). After five minutes ofstirring, Boc protected neomycin isothiocyanate (4) (27.9 mg, 0.02 mmol)was added and the reaction mixture was stirred at room temperatureovernight. Volatiles were evaporated and the crude product was purifiedby column chromatography using DCM-MeOH (0-15% MeOH in DCM) as eluent toafford desired compound Boc protected 88 as off white solid (13 mg,55%); [R_(f) 0.31, 10% EtOH in DCM (v/v)]; IR (KBr, cm⁻¹) 3350-3500(br), 2972, 2109 (br, —C═S), 1701, 1625; ¹H NMR (500 MHz, CD₃COCD₃) δ8.70-8.90 (m, 4H, aromatic hydrogens from naphthalenediimine), 6.40-6.50(m, 2H, NH_(6IV)), 6.05-6.30 (8H, NH_(1I), NH_(6II), NH_(1I), NH_(6II)),5.90-6.05 (br, s, 2H, NH_(2II)), 5.12-5.24 (m, 4H), 4.95-5.10 (m, 4H),4.45 (br, s, 2H), 4.15-4.30 (m, 24H), 3.98-4.06 (m, 4H), 3.87-3.93 (m,4H), 3.40-3.86 (m, 36H), 3.21-3.35 (m, 8H), 2.70-2.80 (m, 4H), 1.80-1.85(m, 4H), 1.10-1.65 (m, 112H, 12×Boc, linker protons); MS (MALDI-TOF) m/zcalcd. for C₁₄₃H₂₃₄N₂₁O₅₀S₅ (M+Na⁺), 3322.93. found 3323.41; UV(Acetone): λ_(max)=378 nm.

The Boc protected compound from above step was taken up in DCM-TFAsolution (2.2 mL, 1:0.1 v/v) and stirred at room temperature underdarkness for 2 h. Progress of reaction was checked by TLC. To this,deionized water (2.0 mL) was added and the mixture was washed with DCM(2×3 mL). The aqueous layer was lyophillized to afford the desiredcompound as slightly greenish white solid (12.84 mg, 91%); ¹H NMR (500MHz, D₂O) δ 8.63-8.69 (m, 4H, aromatic hydrogens fromNaphthalenediimide), 6.05-6.10 (m, 2H), 5.32-5.38 (m, 2H), 5.20-5.24(br, s, 2H), 4.49 (t, J=5.36 Hz, 2H), 4.35-4.45 (m, 2H), 4.26-4.32 (m,2H), 4.21-4.26 (m, 4H), 4.09-4.21 (m, 10H), 3.99 (t, J=10.25 Hz, 2H),3.90 (t, J=9.14 Hz, 2H), 3.32-3.38 (m, 2H), 3.71-3.76 (m, 2H), 3.67 (t,J=10.24 Hz, 2H), 3.43-3.55 (m, 10H), 3.23-3.43 (m, 16H), 3.04-3.16 (m,4H), 2.67-2.84 (m, 4H,), 2.33-2.41 (m, 2H, H_(2Ieq.)), 1.91-2.06 (m, 6H,H_(2Iax.), linker protons from spermidine). MS MALDI-TOF m/z forC₈₆H₁₄₈N₂₂O₂₈S₅ [M+H₂O]⁺, calcd 2116.57. found 2117.213; UV (H₂O):λ_(max)=383 nm, 8383=16811 M⁻¹ cm⁻¹.

Synthesis of Neomycin-Dimer Fluorescein Conjugate (79)

The amino fictionalized neomycin dimer 53 was reacted withfluorescein-isothiocyanate 41b to synthesize Boc protectedneomycin-dimer-fluorescein conjugate (Scheme 24). The amino groups weredeprotected using trifluoroacetic acid to give the desiredneomycin-dimer fluorescein conjugate 79 as their trifluoroacetate salt.

Synthesis of Neomycin Dimer-Fluorescein Conjugate (79)

To a solution of neomycin dimer amine 53 (22.0 mg, 9.0 μmol) in dry DMF(2.0 mL), fluorescein isothiocyanate 41b (4.0 mg, 10.0 μmol) was addedfollowed by addition of triethylamine (2.0 mg, 20.0 mμmol). The reactionmixture was stirred at room temperature under argon atmospheres. Thestirring was continued overnight and the progress of the reaction wasmonitored by TLC. The solvents were evaporated under reduced pressure.The crude product was purified using column chromatography on silica gelusing dichloromethane-methanol as eluent (0 to 20% MeOH in DCM (v/v)).The desired compound was obtained as orange solid (16.5 mg, 68%):R_(f)=0.41 [12% MeOH in DCM (v/v)]; ¹H NMR (500 MHz, CD₃COCD₃) δ 9.20(br, s, 1H), 7.73-7.80 (m, 4H), 7.64-7.70 (m, 2H), 7.10-7.20 (m, 2H),6.63-6.78 (m, 7H), 6.40-6.50 (m, 2H, NH_(6IV)), 6.20-6.28 (m, 4H,NH_(6II), NH_(1I)), 5.90-6.14 (m, 8H, NH_(3I), NH_(2IV), and NH_(2II)),5.27 (m, 2H), 5.21 (m, 2H), 5.01-5.08 (m, 4H), 4.75 (m, 2H), 4.40-4.70(m, 16H), 4.20-4.30 (m, 16H), 4.05 (m, 4H), 3.75-3.98 (m, 16H),3.40-3.70 (m, 20H), 3.20-3.35 (m, 8H), 2.95-3.07 (m, 6H), 1.83-1.88 (p,2H), 1.65-1.78 (m, 8H, linker protons from spermidine), 1.30-1.58 (m,110H, H_(2Ieq), 6×Boc).

To a solution of N-Boc neomycin dimer-fluorescein (15.0 mg, 50.0 μmol)in dioxane (2.0 mL), 4 N HCl in dioxane (1.0 mL) was added and thereaction mixture stirred for 15 min. A yellow precipitate was formedafter stirring the reaction mixture for 20 min. The reaction mixture waswashed with diethyl ether/hexane [3×2.0 mL, 1:1 (v/v)]. The precipitatewas dissolved in water and then it was lyophilized to give the desiredproduct as an orange solid (11.3 mg, 90%): ¹H NMR (500 MHz, CD₃COCD₃) δ8.61-8.67 (m, 1H), 8.10-8.20 (m, 1H), 7.72 (m, 1H), 7.23-7.32 (m, 3H),7.10-7.20 (m, 3H), 7.02-7.10 (m, 1H), 5.30-5.50 (m, 2H), 5.10-5.30 (m,2H), 4.30-4.50 (m, 4H), 4.20-4.30 (m, 4H), 4.10-4.20 (m, 6H), 3.75-4.04(m, 10H), 3.60-3.80 (m, 12H), 3.25-3.60 (m, 24H), 3.02-3.20 (m, 10H),2.70-2.98 (m, 4H), 2.35-2.50 (m, 2H, H_(I2eq)), 1.80-1.98 (m, 2H,H_(I2ax)), 1.50-1.60 (m, 2H), 1.10-1.40 (m, 6H); MS (MALDI-TOF) m/zcalcd. For C₈₇H₁₄₃N₁₉O₂₉S₅(M+H₂O⁺) 2077.89. found 2095.13 [M+H₂O]⁺.

Synthesis of Neomycin-Boc Dimer with Azide Functionalization (86)

Neomycin dimers were synthesized by coupling a bisazide derivative withan alkyne terminated neomycin derivative. In the first step, aderivatized bisazido linker was synthesized in a two-step reaction(scheme 25). Bis(2-chloroethyl)amine was reacted with excess sodiumazide at elevated temperature and converted intobis(2-azidooethyl)amine. The bis(2-azidooethyl)amine was then reactedwith 6-bromo hexanoyl chloride under basic conditions to form a bisazide83 that a bromo ended appendage from the middle of linker in highyields.

Neomycin dimer with a bromo functionalized dimer 85 was synthesized byreacting Hexa-N-Boc deoxy-neomycin-5″-alkyne 84 (2 mole equival.) withN,N-bis(2-azidoethyl)-6-bromohexanamide (1 mole equival.) using clickchemistry (scheme 26). The bromide functionality on the neomycin dimerwas substituted to an azido group via reaction with sodium azide. Theazido ended neomycin dimer 84 can be reduced to the amine or directlycoupled to fluorescent alkynes used to synthesize fluorescent neomycindimer conjugates for the high throughput probing of nucleic acidbinding.

Synthesis of an Azido Functionalized Dimer for Conjugation toFluorophores Via Click Chemistry Synthesis of Bis(2-Azidoethyl)Amine(82)

To a solution of bis(chloroethyl) amine hydrochloride (1.4 gm, 1.0 mmol)in water (10.0 mL), sodium azide (3.2 gm, 5.0 mmol) was added and thereaction mixture was stirred at 100° C. for 48 h. The reaction mixturewas allowed to come to room temperature. It was followed by addition of3M NaOH until the pH of the solution was 10. The reaction mixture wasextracted with diethyl ether (3×20.0 mL). The organic layer was driedover Na₂SO₄. Removal of volatiles gave the desired compound as greenishcolored oil (1.4 g, 90%): R_(f)=0.30[15% CH₃OH in CH₂C12 v/v)]; IR(neat, cm⁻¹) 3411 (broad), 2976, 2104 (—N₃), 1618, 1541; ¹H NMR (500MHz, CDCl₃) δ 3.35 (t, J=6.62 Hz, 4H, N₃—CH₂—CH₂—N—), 2.85 (t, J=6.52Hz, 4H, N₃—CH₂—CH₂—N—); ¹³C NMR (500 MHz, CDCl₃): δ 51.27(N₃—CH₂—CH₂—N—), 48.04 (N₃—CH₂—CH₂—N—).

Synthesis of N, N-Bis(2-Azidoethyl)-6-Bromohexanamide (83)

To a solution of bis (azido diethyl) amine (0.5 gm, 0.3 mmol) indichloromethane (2.0 mL), an aqueous solution of 3M NaOH (2.6 mL) wasadded and the reaction mixture stirred at 0° C. for 15 min followed byaddition of a solution of 6-bromohexanoyl chloride (1.3 gm, 0.6 mmol in1.0 mL dichloromethane) dropwise. The reaction mixture was stirred for 1h at 0° C. The organic layer was washed with 3N HCl (3×3.0 mL), driedover Na₂SO₄ and evaporation of solvent results in greenish oil (0.9 gm,86%): R_(f)=0.5 [50% ethylacetate in hexane (v/v)]; IR (neat, cm⁻¹) 3340(broad), 2914, 2104 (broad, N₃), 1701, 1465; ¹H NMR (500 MHz, CDCl₃) δ3.42-3.50 (m, 6H), 3.37-3.41 (m, 2H), 3.34 (t, J=6.62 Hz, 2H), 2.30 (t,J=7.41 Hz, 2H), 1.80 (p, J=6.93 Hz, 2H), 1.59 (p, J=7.72 Hz, 2H), 1.40(p, J=7.41 Hz, 2H); ¹³C NMR (500 MHz, CDCl₃) δ 173.26, 49.91, 49.45,48.11, 46.21, 33.72, 32.79, 32.52, 27.79, 24.11.

Synthesis of Neomycin Clickable Dimer Br-End (85)

To a solution of Hexa-N-Boc deoxy-neomycin-5″-propargyl ether (43.0 mg,14.0 μmol) in EtOH (2.0 mL), CuSO₄ (0.5 mg, 3.5 μmol, in 0.2 mL water),and sodium ascorbate (1.4 mg, 7.0 μmol, in 0.2 mL water) was added andthe reaction mixture was stirred at room temperature for 15 min. N,N-bis(2-azidoethyl)-6-bromohexanamide (2.3 mg, 7.0 μmol) in EtOH (0.2mL) was added dropwise and the reaction mixture was stirred vigorouslyat room temperature. The progress of the reaction was monitored by TLC.The crude product was purified on a silica gel column usingdichloromethane-ethanol as eluent which yielded the desired product asyellowish solid (36.2 mg, 80%):R_(f)=0.38 [0 to 10% EtOH in DCM (v/v)];¹H NMR (500 MHz, CD₃COCD₃): δ 8.21-8.34 (m, 2H, triazole), 8.04-8.15 (m,2H, triazole), 6.52-6.67 (br, s, 2H, NH_(6IV)), 6.30-6.41 (br, s, 2H),5.96-6.26 (m, 10H, NH_(6II), NH_(1I), NH_(3I), NH_(2IV), and NH_(2II)),5.20-5.31 (m, 4H), 5.03-5.08 (m, 2H), 4.89-5.02 (m, 4H), 4.85 (d, J=8.35Hz, 2H), 4.67-4.81 (m, 18H), 4.50-4.57 (br, s, 2H), 4.23-4.41 (m, 12H),4.05 (s, 4H), 3.78-3.96 (m, 10H), 3.53-3.74 (m, 20H), 3.38-3.55 (m,10H), 3.14-3.36 (m, 8H), 1.82 (p, J=6.94 Hz, 2H), 1.33-1.65 (m, 114H,H_(2Ieq), 6×Boc, linker protons); MS (MALDI-TOF) m/z calcd. forC₁₂₈H₂₁₆BrN₂₅O₅₁ (M+H₂O⁺), 3016.40. found 3016.40.

Synthesis of Neomycin Clickable Dimer Azide End (86)

To a solution of 85 (100.0 mg, 3.3 μmol) in DMF (2.0 mL), NaN₃ (100.0 mg1.5 mmol) was added and the reaction mixture was stirred at 100° C. for14 h. The volatiles were removed under reduced pressure. The reactionmixture was dissolved in ethyl acetate (5.0 m) and then it was washedwith water (3×5.0 m). The organic layer was dried over Na₂SO₄. Removalof volatiles under reduced pressure results in a brownish solid (93.8mg, 95%). The product mixture was used in next step without furtherpurification: IR (neat, cm⁻¹) 3300-3500 (broad), 2110 (—N₃), 1610-1650,1505; ¹H NMR (500 MHz, CD₃COCD₃) δ 8.20-8.31 (m, 2H, triazole),8.06-8.16 (n, 2H, triazole), 6.55-6.69 (br, s, 2H, NH_(6IV)), 6.28-6.42(br, s, 2H), 5.95-6.27 (m, 10H, NH_(6II), NH_(1I), NH_(3I), NH_(2IV),and NH_(2II)), 5.20-5.31 (m, 4H), 5.07-5.11 (m, 2H), 4.85-5.04 (m, 6H),4.55-4.83 (m, 20H), 4.20-4.50 (m, 12H), 4.05 (s, 4H), 3.80-3.95 (m,10H), 3.53-3.77 (m, 20H), 3.35-3.53 (m, 10H), 3.15-3.37 (m, 8H),1.33-1.65 (m, 116H, H_(2Ieq), 6×Boc, linker protons); MS (MALDI-TOF) m/zcalcd. for C₁₂₈H₂₁₆N₂₈O₅₁ (M+H₂O⁺), 2979.50. found 2980.10.

Synthesis of Alkyne Functionalized Neomycin (89)

A short alkyne free of triazole ring can also be prepared by reaction ofBoc protected neomycin amine 6 with propargyl chloroformate (Scheme 27).This leads to the formation of a carbamate bond. The alkyne can then beused towards click chemistry reactions.

Synthesis of Hexa-N-Boc deoxy-neomycin-5″-propargyl alkyne (7)

To a solution of Hexa-N-Boc deoxy-neomycin-5″-amine (60.00 mg, 0.05mmol) in dry DCM (10.0 mL), triethylamine (10.0 mg, 0.1 mmol) was addedfollowed by propargyl chloroformate (13.7 mg, 0.1 mmol) at −78° C. underthe atmosphere of argon. The reaction mixture was stirred for 6 h andthe progress of the reaction was monitored using TLC. The disappearanceof starting material suggested completion of reaction. The reactionmixture was washed with water (10 mL) and the organic layer was driedover Na₂SO₄. The crude reaction mixture was purified using columnchromatography on a silica gel column using dichloromethane-ethanol aseluent (0 to 10% EtOH in DCM). The desired product was obtained as whitesolid (46.0 mg, 72%): R_(f)=0.48 in 10% EtOH in dichloromethane (v/v)];IR (KBr, cm⁻¹) 3300-3400 (br), 2960, 2910, 2112 (br, alkyne), 1712,1608, 1450; ¹H NMR (500 MHz, CD₃COCD₃) δ 7.01 (s, 1H), 6.62 (s, 1H),6.20-6.30 (m, 2H, NH_(6IV) and NH_(1I) or NH_(6II)), 6.12 (m, 1H,NH_(1I) or NH_(6II)), 6.01-6.08 (m, 2H, NH_(3I), NH_(2IV)), 5.92 (s, 1H,NH_(2II)), 5.10-5.20 (m, 3H), 4.92 (s, 3H), 4.30-4.40 (m, 2H), 4.10-4.25(m, 4H), 4.01-4.09 (m, 4H), 3.95 (t, J 6.02 Hz, 2H), 3.70-3.87 (m, 6H),3.35-3.70 (m, 18H), 3.30-3.40 (s, 4H), 2.97 (s, 1H), 1.20-1.65 (m, 56H,H_(2Iax), 6×Boc); MS (MALDI-TOF) m/z calcd. for C₅₇H₉₇N₇O₂₆ [M+Na⁺],1296.41, obsd: 1317.29 [M+H₂O+2H]⁺.

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1-27. (canceled)
 28. A nucleic acid probe comprising thiazole orange, alinker moiety having a first end and a second end, and a nucleic acidbinding moiety, wherein the thiazole orange is covalently attached tothe first end of the linker moiety and the second end of the linkermoiety is covalently attached to the nucleic acid binding moiety,wherein the nucleic acid binding moiety comprises an aminoglycosidemoiety comprising unbound primary, secondary, and/or guanidino aminogroup(s) available for nucleic acid binding, and wherein the linkermoiety is covalently attached to the nucleic acid binding moiety througha hydroxyl group of the aminoglycoside moiety.
 29. The probe of claim28, wherein the aminoglycoside moiety is amikacin, apramycin, arbekacin,bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin,geneticin, gentamicin, isepamicin, kanamycin, lividomycin, micronomicin,neamine, neomycin, netilmicin, paromomycin, ribostamycin, sisomicin,spectinomycin, streptomycin, streptonicozid, tobramycin, trospectomycin,viomycin, an analog thereof, a derivative thereof, a homo dimer thereof,a heterodimer thereof or pharmaceutically acceptable salt thereof. 30.The probe of claim 28, wherein the aminoglycoside moiety is neomycin, ahomo dimer thereof, or a hetero dimer thereof.
 31. The probe of claim28, wherein the nucleic acid binding moiety further comprises asecondary binding moiety linked to the aminoglycoside moiety.
 32. Theprobe of claim 31, wherein the secondary binding moiety is a minorgroove binder.
 33. The probe of claim 31, wherein the secondary bindingmoiety is Hoechst 33258, Hoechst 33342, Hoechst 34580, distamycin A,netropsin, 4′,6-diamidino-2-phenylindole (DAPI),4,4′-(1-Triazene-1,3-diyl)bis(benzenecarboximidamide), lexitropsin,4-[(3-Methyl-6-(benzothiazol-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-pyridiniumiodide (BEBO), or BOXTO(4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinoliniumchloride).
 34. The probe of claim 28, wherein the linker moiety is oneof

where Y is


35. The probe of claim 28, wherein the linker moiety is

where X is O, S, or NH, and Y is O, S, or NH.
 36. The probe of claim 28,wherein the linker moiety is -(L₁)_(v)-, wherein v is independently1-20, and wherein each (L₁) is independently O, N, S, C₁-C₈ alkyl, C₂-C₈alkenyl, C₂-C₈ alkoxy, aryl, heteroaryl, heterocyclyl,


37. The probe of claim 28, wherein the linker moiety is O, N, S, C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkoxy,


38. The probe of claim 28, wherein a fluorescence of the probe changesupon binding of the probe to a nucleic acid.
 39. The probe of claim 28,wherein the nucleic acid binding moiety binds to RNA, A-DNA, B-DNA,double stranded DNA, triple stranded DNA, or quadruplex DNA.
 40. Theprobe of claim 39, wherein the nucleic acid binding moiety binds to amajor groove of the DNA.
 41. The probe of claim 29, wherein the neomycinis neomycin B or neomycin C.
 42. The probe of claim 29, wherein thegentamicin is gentamicin A, gentamicin C1, gentamicin C1a, or gentamicinC2.
 43. The probe of claim 29, wherein the kanamycin is kanamycin A,kanamycin B, or kanamycin C.
 44. The probe of claim 39, wherein thenucleic acid binding moiety binds to a major groove of the doublestranded DNA.
 45. The probe of claim 39, wherein the double stranded DNAis A-DNA.
 46. The probe of claim 39, wherein the double stranded DNA isB-DNA.
 47. The probe of claim 28, wherein the linker moiety iscovalently attached to the 5′ OH position of the ribose ring of neomycinB.